Treatment and prevention of Gardnerella vaginalis infections

ABSTRACT

The present invention is drawn to the nucleic and amino acid sequences encoding vaginolysin (VLY) toxin from  Gardnerella vaginalis , and biologically active fragments and variants thereof. The invention is also directed to anti-VLY antibodies and to their use therapeutically and in a new ELISA assay of VLY toxin. Other embodiments of the invention are directed to VLY toxoids and to vaccines that use the new VLY toxoids as immunogens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present application is a divisional of application Ser. No.12/922,837, filed Sep. 15, 2010, under 35 U.S.C. §120, itself a nationalstage application under 35 U.S.C. §371 of PCT Application No.PCT/US2009/037328, filed Mar. 16, 2009; and claims priority of U.S.Provisional Application No. 61/036,943, filed Mar. 15, 2008, and U.S.Provisional Application No. 61/057,190, filed May 29, 2008, the entirecontents of which are hereby incorporated by reference as if fully setforth herein, under 35 U.S.C. §119(e).

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant AI065450awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the discovery of the nucleic acidsequence and amino acid sequence encoding a newly identified G.vaginalis pore-forming toxin called vaginolysin (VLY), and to methodsfor diagnosing, preventing and treating G. vaginalis and bacterialvaginosis (BV). Other aspects are related to vaccines that include atoxoid form of VLY as an immunogen.

2. Description of the Related Art

Bacterial vaginosis (BV) is the most common vaginal infection worldwideand is associated with significant adverse consequences includingpreterm labor and delivery (40, 41), post-partum endometritis (42), andan increased risk of HIV acquisition (43-45). Reported prevalence ratesrange from 10-40% depending upon the population studied (46). However,suboptimal methods of diagnosis and a high percentage of asymptomaticpatients make the true prevalence of BV difficult to ascertain.Gardnerella vaginalis is a bacterial species associated with bacterialvaginosis (BV).

The pathogenesis of BV remains poorly understood. It is most commonlydefined as a pathological state characterized by the loss of normalvaginal flora, particularly Lactobacillus species, and overgrowth ofother microbes including Gardnerella vaginalis, Bactericides species,Mobiluncus species, and Mycoplasma hominis. Recent data however, suggesta primary role for G. vaginalis as a specific and sexually transmittedetiological agent in BV, as was initially postulated by Gardner andDukes in 1955 (47-49).

Alterations of both local host immunity and the genital tract microfloraappear to contribute to the pathogenesis of BV (39), which can bedifficult to eradicate even using targeted antimicrobial therapy (4). Inaddition, randomized trials of antibiotics for the prevention ofBV-associated preterm birth have not shown consistently beneficialeffects, suggesting that host inflammatory responses set in motion earlyin the course of disease may contribute significantly to theconsequences of infection (26, 27).

In the 1950s, Leopold (25) and then Gardner and Dukes (14) observedabundant small, pleomorphic gram-variable rods in the genital tract ofwomen with BV. This organism, first called Haemophilus vaginalis (13)and repeatedly renamed as more information about its characteristicsbecame available (reviewed in (5)), is now classified as Gardnerellavaginalis, the sole member of the genus Gardnerella (16, 30).Phylogenetic analysis based on 16S rRNA places Gardnerella in thegram-positive family Bifidobacteriales. An abundance of G. vaginalis anda paucity of Lactobacillus species are characteristic of a BV-associatedmicroflora, but the relative contribution of G. vaginalis to thepathogenesis of BV is not clear. G. vaginalis is present in essentiallyall cases of BV but can also be detected in a minority of asymptomaticwomen (1) Likewise, several groups have demonstrated that the vaginalmicroflora is exceedingly complex in BV where the vaginal mucosa is hostto many non-Gardnerella organisms (12, 18, 20). Mechanistic studies ofBV and its adverse consequences have been limited by the absence ofdefinitive diagnostic testing and a suitable animal model (22, 23, 26).Existing methods of diagnosis for BV are suboptimal and frequentlyunderutilized by practitioners. A recent study by Hogan et al. reportsthat the prevalence of BV among pregnant women varies greatly dependingon the diagnostic criteria used (51). Established in 1983, Amsel'scriteria are widely accepted as the best available means for diagnosingBV in the clinical setting, however, these criteria are complex,somewhat subjective, and necessitate that microscopy equipment bepresent on site (52, 53). The Nugent scoring system for interpretationof gram-stained vaginal smears was put forth in an attempt tostandardize diagnosis of BV and increase inter-rater reliability (54).While the Nugent scoring system exhibits superior sensitivity andspecificity compared to the Amsel criteria (55), its use remains largelyrestricted to research protocols. Furthermore, questions regarding therisk of potential morbidities and the need for antimicrobial therapy inthose women found to have “intermediate flora” remain unanswered (56,57).

Several alternative diagnostic methodologies focusing upon the detectionof microbial virulence factors produced by the various BV-associatedorganisms have been proposed in recent years. These include detection ofbacterial sialidases, determination of amine levels, and measurement ofproline aminopeptidase activity (58-60). While these techniques arerelatively simple, rapid and inexpensive, they fail to identify thespecific microbial pathogens present. A potential role for novel,molecular-based techniques for diagnosing BV has recently emerged.Importantly, preliminary studies evaluating these PCR-based strategieshave provided additional evidence for G. vaginalis as the primaryetiologic agent of BV (61-63). Menard et al. analyzed 213 vaginalsamples from pregnant women using molecular probes targeting 8BV-related organisms (64). These authors report that an increased loadof G. vaginalis (>10⁹ copies of G. vaginalis DNA per ml) had both highnegative and positive predictive values for the diagnosis of BV. Whilethese molecular based diagnostic strategies are promising, the requiredexpertise, laboratory resources and expense limits their use in theprimary care setting.

G. vaginalis produces a cholesterol-dependent cytolysin (CDC) (proteinpore-forming toxin) called vaginolysin (“VLY”) that acts as a hemolysin(8, 35, 50). IgA-mediated immune responses to the hemolysin occur duringBV and are useful as a marker of disease (8, 35). However, completecharacterization of the VLY has been limited by the absence of geneticinformation and an inability to produce recombinant toxin. Thereforethere is a great need to sequence and characterize the human-specificVLY toxin, and for methods for treating or preventing G. vaginalis andBV.

The global impact of the HIV epidemic cannot be overstated. Even asantiretroviral drugs prolong and improve life for HIV-infected people inwealthy nations, millions of people suffer and ultimately perish fromthe ravages of the disease worldwide. Despite this, and despite decadesof research, prevention and cure of HIV remain elusive goals. Thus,there is a need for novel and creative approaches to preventing HIVacquisition. BV is exceedingly common, especially in Africa, where morethan 50% of women in numerous trials, including the recent trial ofacyclovir for HSV suppression in Tanzania (1), were infected with BV. BVhas been repeatedly associated with both increased risk of HIVacquisition and increased viral shedding among those already infectedwith HIV2. In vitro, treatment of HIV-infected cells with Gardnerellaleads to increased production of viral transcripts. Comparatively littleattention has been paid to targeting BV as a means of affecting theprogression of the HIV epidemic due to several factors: (1) BV is not a“traditional” sexually transmitted infection (STI) and is often omittedfrom analyses of STI-HIV interactions; (2) Gardnerella is enigmatic:difficult to culture, without an available genome sequence, lackingknown virulence factors, and (perhaps most importantly) without ananimal model; and (3) BV and Gardnerella colonization are extremelydifficult to eradicate even with targeted antibiotic therapy. Arandomized, controlled trial of mass antibiotic treatment targeting STIin Uganda did not affect the prevalence of BV in either the control orthe treatment group, emphasizing the challenges of BV prevention andtreatment (and failing to address the impact of an efficacious BVtherapy) (4).

Therefore there is a great need for a new methods and compositions to G.vaginalis and BV to minimize the risk of transmitting HIV from person toperson, particularly from an HIV-positive mother who has G. vaginalis toa fetus or an infant during delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the drawings.

FIG. 1: Phylogenetic relationship between VLY and other members of thecholesterol-dependent cytolysin (CDC) family. (A) Phylogram offull-length CDC protein sequences predicted by the neighbor joiningalgorithm. Numbers represent calculated relative phylogenetic distances.(B) Multiple alignments of undecapeptide regions from known CDC. Thepredicted amino acid sequence of VLY contains a variant undecapeptideregion most similar to the undecapeptide from intermedilysin (ILY). Thesequence labeled “consensus” corresponds to the undecapeptide from MLY,PLY, SUI, IVN, ALV, SPH, THU, SLO, ALO, LLO, PFO, CER, and TET. (C)Western blot of lysed G. vaginalis bacteria (G. vaginalis) and purified,recombinant VLY, probed with anti-PLY monoclonal antibody. Abbreviationsfor CDC proteins: PLY, pneumolysin; LLO, listeriolysin O; IVN,ivanolysin; SLG, seeligeriolysin; SPH, sphaericolysin; ALO, anthrolysinO; CER, cereolysin; PFO, perfringolysin O; ALV, alveolysin; TET,tetanolysin O; PYO, pyolysin; MLY, mitilysin; SLO, streptolysin O; SUI,suilysin; THU, thuringensolysin.

FIG. 2: Human-specific, cholesterol-dependent hemolytic activity ofvaginolysin (VLY). (A) Washed human (hRBC) or sheep (sRBC) erythrocytes(1% solution in PBS) were exposed to the indicated concentrations ofpurified recombinant VLY for 30 min, followed by pelleting of cells.Hemoglobin release was measured by OD₄₁₅ of the supernatant andnormalized to 100% lysis for each species tested (P<0.01, ANOVA). (B)Erythrocytes from various species were exposed to VLY or thenon-species-specific toxin pneumolysin (PLY; both toxins at 5 μg/ml) andlysis measured. (C) Addition of cholesterol (Ch) at 1 μg/ml or 10 μg/mlinhibits human erythrocyte lysis by VLY (5 μg/ml) (P<0.001, ANOVA).

FIG. 3: Host specificity of VLY depends on the complement regulatorymolecule CD59. (A) VLY-induced lysis of human erythrocytes was inhibitedby monoclonal antibody to human CD59 (P<0.0001) but not antibody toanother GPI-anchored cell surface antigen (CD55) or mock treatment(PBS). (B) Antibody to CD59 does not inhibit PLY-mediated lysis of humanerythrocytes. (C) LDH release from Chinese hamster ovary (CHO) cellstransfected with empty vector (IRES) or human CD59 (IRES-hCD59) andexposed to VLY (10 μg/ml) for 30 min. Transfection of human CD59increases VLY-mediated lysis (P<0.0001). (D) Transfection of human CD59into CHO cells does not affect PLY (1 μg/ml)—mediated lysis (P>0.05).

FIG. 4: VLY-mediated epithelial cell activation and erythrocyte lysisrequire P480. (A) Human cervical epithelial cell line HeLa was treatedfor 30 min with media alone (-), VLY or VLY (P480W) (1-10 μg/ml) priorto lysis and Western blotting with antibodies specific for total (p38)and phospho-p38 (pp38) MAPK. (B) HeLa cells were treated with VLY or VLY(P480W) (10 μg/ml) for 2 hr prior to RNA extraction and assay ofrelative quantity of interleukin-8 (IL-8) message by real-time PCR. (C)Human (hRBC) and sheep (sRBC) erythrocytes were treated with theindicated concentrations of VLY or VLY (P480W) and hemolysis assessed asabove. (D) Human (hRBC) erythrocytes were treated with the indicatedconcentrations of pneumolysin (PLY) or PLY (W435P) and hemolysisassessed as above.

FIG. 5: Novel antibody techniques for the detection of VLY. (A) Westernblot of G. vaginalis 14018 lysate probed with rabbit polyclonalantiserum (1:500,000 dilution). Numbers represent approximate MW in kD(B) Immunofluorescent detection of VLY production by G. vaginalis usingpre-immune rabbit serum (left panel) or anti-VLY antiserum (rightpanel). Anti-rabbit IgG-AF488 was the secondary antibody (green). DNAstaining with DAPI demonstrates bacteria in both panels (blue). Scalebar: 10 μm.

FIG. 6: Quantification of VLY production in vivo. (A) Detection of VLYin G. vaginalis supernatants by ELISA at various time points followinginoculation of broth culture. (B) Bacterial growth (OD600) over thecourse of the experiment.

FIG. 7: Polyclonal immune serum inhibits VLY-mediated hemolysis. (A)Human erythrocytes were exposed to varying concentrations of purifiedrecombinant VLY for 30 min. Cells were pelleted, and hemoglobin releasewas determined by OD415 of the supernatant. Values were normalized to100% lysis. When indicated, VLY was preincubated with pre-bleed (VLY+PB)or immune serum (VLY+IS) diluted 1:50 for 30 min prior to use in theassay. (B) Erythrocytes were exposed to VLY (500 ng/ml), VLY+PB, orserial dilutions of VLY+IS. (P<0.001)

FIG. 8: Immune serum inhibits VLY-mediated lysis of human cervical andvaginal cells. Human cervical (A, HeLa) or vaginal (B, VK2) epithelialcells were exposed to VLY (10 μg/ml), VLY+PB, or VLY+IS. Lysis wasmeasured by LDH release assay following 30 min of incubation with toxin.Values were normalized to 100% lysis for each cell line (P<0.005).

SUMMARY OF THE INVENTION

Certain embodiments of the present invention are directed to an isolatednucleic acid encoding VLY toxin protein from G. vaginalis, identified asthe nucleotide sequence SEQ ID No: 1 from strains 14018 or 14019, SEQ IDNO: 3 from strain 49145, or SEQ ID NO: 10 from strain ARG3, anddegenerate variants or fragments thereof. Other embodiments are directedto an isolated nucleic acid encoding domain 4 of VLY from G. vaginalis,identified as nucleotide sequence of SEQ ID No: 4 from strains 14018 or14019 or SEQ ID NO: 12 from strain 49145, and degenerate variants orfragments thereof. Other embodiments are directed to an isolated nucleicacid encoding the undecapeptide of VLY from G. vaginalis, identified asnucleotide sequence of SEQ ID No: 6, from strains 14018 or 14019 or SEQID NO: 14 from strain 49145, and degenerate variants or fragmentsthereof.

Other embodiments are directed to isolated and purified VLY protein fromG. vaginalis, identified by the amino acid sequence set forth in SEQ IDNO: 2 from strains 14018 and 14019 and SEQ ID NO: 11 from strain ARG3,and biologically active fragments or variants thereof. Other embodimentsare directed to the isolated and purified conserved undecapeptide regionof VLY of G. vaginalis, the amino acid sequence (EKTGLVWEPWR) of whichis set forth as SEQ ID NO: 7, or a biologically active fragment orvariant thereof, and the undecapeptide region for toxoid 480P(EKTGLVWEWWR) set forth in SEQ ID NO: 8. Also claimed are isolated andpurified domain 4 peptides of VLY of G. vaginalis, the amino acidsequences of which are set forth in SEQ ID NO: 5 from strains 14018 and14019, or SEQ ID NO: 13 from strain 49145, and biologically activefragments or variants thereof. Another embodiment is directed toisolated and purified immunogenic G. vaginalis VLY polypeptidefragments, that include at least ten consecutive amino acid residues ofSEQ ID NO: 2 from strains 14018 or 14019, and SEQ ID NO: 3 from strain49145, and variants thereof.

Other embodiments are directed to various isolated and purifiedpore-forming toxoids of VLY from G. vaginalis, as defined herein, and totheir use in a VLY vaccine.

Other embodiments are directed to a method for diagnosing and treating aG. vaginalis infection or bacterial vaginosis in a patient, the steps ofwhich include (a) obtaining a biological sample from the patient, (b)detecting the presence of VLY protein having the amino acid sequence setforth in SEQ ID NO: 2 or SEQ ID NO: 3, or a biologically active fragmentor variant thereof in the biological sample taken from the patient, and(c) if the VLY protein is detected then administering to the patient atherapeutically effective amount of an antibiotic known to treat G.vaginalis or a protective agent that is a member selected from the groupcomprising anti-VLY antibody, anti-PLY antibody, anti-CD59 antibody,anti-pneumolysin antibody, soluble CD59, G. vaginalis VLY toxoid, or abiologically active fragment or variant thereof.

Other embodiments are directed to methods for treating or preventing aG. vaginalis infection or bacterial vaginosis in a patient, byadministering to the patient a therapeutically effective amount of aprotective agent that is a member selected from the group comprisinganti-VLY antibodies including newly discovered rabbit polyAnti-VLY,anti-PLY antibodies, anti-CD59 antibodies, anti-pneumolysin antibodies,soluble CD59, G. vaginalis VLY toxoid, or biologically active fragmentsor variants thereof.

Other embodiments are directed to compositions for therapeutic use thatinclude anti-VLY antibodies, preferably polyAnti-VLY, and tocompositions that include two compounds selected from the groupcomprising soluble CD59, anti CD59, anti-VLY, anti-PLY oranti-pneumolysin monoclonal or polyclonal antibodies or immunologicallyactive fragments or variants thereof including polyAnti-VLY, VLY toxoidsor immunologically active fragments or variants thereof, and antibioticsknown to treat bacterial vaginosis or G. vaginalis.

Other embodiments are directed to an ELISA kit for diagnosing G.vaginalis infection in a patient, comprising a detection antibody thatspecifically binds to VLY protein identified by SEQ ID NO: 2 or SEQ IDNO: 11, or a biologically active fragment thereof, including a kitwherein the detection antibody is rabbit polyAnti-VLY. In someembodiments the detection antibody is covalently bound to an enzyme. Thekit optionally provides a substrate for the enzyme. Other embodimentsare directed to an ELISA kit that also contains a secondary antibodycovalently bound to an enzyme, which secondary antibody specificallybinds to the detection antibody, and a substrate for the enzyme. Stillother embodiments are directed to an Elisa kit including a capturemolecule that specifically binds to VLY, including soluble CD59,anti-VLY antibody, anti-PLY antibody, and anti-pneumolysin.

Other embodiments are directed to methods for reducing or preventing thetransmission of HIV to another human by a woman infected with HIV andbacterial vaginosis, by administering to the woman before the womanengages in a sexual activity, a therapeutically effective amount of acomposition comprising a protective agent selected from the groupcomprising soluble CD59, anti CD59 or anti-VLY, anti-PLY, polyAntiPLY,or anti-pneumolysin monoclonal or polyclonal antibodies, VLY toxoids orbiologically active fragments or variants thereof. Other embodiments aredirected to methods of reducing or preventing maternal to fetaltransmission of HIV from a pregnant woman diagnosed as being infectedwith both HIV and bacterial vaginosis, by administering to the womanprior to a vaginal birth of a fetus, a therapeutically effective amountof a protective agent selected from the group comprising soluble CD59,anti CD59 or anti-VLY, anti-PLY, polyAntiPLY, or anti-pneumolysinmonoclonal or polyclonal antibodies, VLY toxoids or fragments orvariants thereof.

Another embodiment is directed to the polyclonal rabbit anti-VLYantibody poly-Anti-VLY, or an immunologically active fragment or variantthereof, ant to compositions that include this antibody.

DEFINITIONS

By “protein” or “polypeptide” is meant any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation).

A “pure polypeptide” refers to a polypeptide substantially free fromnaturally associated molecules, i.e., it is at least 75% (e.g., at least80, 85, 90, or 95; or 100%) pure by dry weight. Purity can be measuredby any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

“Peptide variants” and “peptide modifications” are used synonymouslyhere and mean polypeptides that may contain one or more substitutions,additions, deletions and/or insertions such that the therapeutic andantigenic properties of the peptide encoded by the variant are notsubstantially diminished, relative to the corresponding peptide such asVLY. Such modifications may be readily introduced using standardmutagenesis techniques, such as oligonucleotide directed site-specificmutagenesis. Variants also include what are sometimes referred to as“fragments.” Peptide variants may contain one or more amino acidsubstitutions, additions, deletions and/or insertions. When VLY toxinand toxoids are discussed in the context of expression, activity,immunogenicity or binding to receptors on target cells, the termsinclude biologically active fragments and variants thereof. The aminoacids used to make peptides and variants include synthetic amino acidsknown in the art.

As used herein, the term VLY means wild type vaginolysin (“VLY”)polypeptide that is the human-specific CDC from G. vaginalis. VLY is theonly known species-specific factor of G. vaginalis. VLY is encoded byDNA SEQ ID NO. 1 in G. vaginalis 14018 and 14019; and by DNA SEQ ID NO.3 in strain 49145. The amino acid sequence of VLY is set forth in SEQ IDNO. 2. As used herein VLY includes biologically active fragments andpeptide variants thereof, whether naturally-occurring mutants orman-made mutants.

As used herein, the term VLY toxoid means VLY protein that has reducedpore-forming activity compared to wild type VLY due to the presence ofone or more amino acid substitutions, but that retains enoughimmunologic activity to elicit an immune response in an animal.

Unless otherwise indicated, a “therapeutically effective amount” of acompound is an amount that provides a therapeutic benefit in thetreatment or management of a disease or condition such as Bacterialvaginosis (BV) or G. vaginalis, delays or minimizes one or more symptomsassociated with the disease or condition, or enhances the therapeuticefficacy of another therapeutic agent. An agent is said to beadministered in a “therapeutically effective amount” if the amountadministered results in a desired change in the physiology of arecipient mammal, (e.g. decreases one or more symptoms of the BV or G.vaginalis, or decreases the amount of G. vaginalis in a biologicalsample taken from the patient to a level that is at least about 10% lessthan the level before drug treatment). A therapeutically effectiveamount for reducing the risk of transmitting HIV from a G.vaginalis-infected woman to a sexual partner or a baby during birth, isan amount that reduces or eliminates viral shedding from HIV-infectedcells or that reduces or eliminates the viral load in a biologicalsample taken from the vagina/birth canal.

Nucleic acids in the context of this invention include“oligonucleotides”, a term that refers to an oligomer or polymer ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid. The termtherefore covers, for example, (a) a DNA which has the sequence of partof a naturally occurring genomic DNA molecule but is not flanked by bothof the coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein.

As used herein, protective agents are agents that block or reduce thetoxic biological activity of VLY. They include agents that bind to orassociate with VLY at a site that interferes with the ability of VLY tobind to CD59 receptor on a target host cell, or otherwise reduce theability of VLY to cause pores in target cells, or reduce the ability ofVLY to increase shedding in HIV-infected cells, or reduce G. vaginalisinfections or BV. Protective agents include soluble CD59 or fragments orvariants thereof that are capable of neutralizing VLY; polyclonal andmonoclonal antibodies to CD59, PLY, VLY, pneumolysin, and immunologicfragments or variants thereof, and VLY toxoids that can compete with themore toxic VLY for binding to CD59. Also included is newly discoveredpolyAnti-VLY and antibiotics that are known to treat bacterial vaginosisand G. vaginalis infections.

DETAILED DESCRIPTION

In the following description, for the purposes of explanation, numerousspecific details are set forth in order to provide a thoroughunderstanding of the present invention. It will be apparent, however, toone skilled in the art that the present invention may be practicedwithout these specific details.

Bacterial vaginosis, a chronic infectious/inflammatory diseaseassociated with preterm birth, is strongly linked with the mucosalovergrowth of G. vaginalis and its attachment to epithelial cells. Tosummarize, we have cloned, sequenced, characterized, isolated andpurified the human-specific cholesterol-dependent cytolysin (CDC)pore-forming toxin vaginolysin (VLY) from several human strains of G.vaginalis (14018, 14019, 49145, and ARG3). VLY, the only knownspecies-specific factor for G. vaginalis, is most closely related tointermedilysin (ILY) from Streptococcus intermedius. (15, 29). We havekept the name “vaginolysin” (VLY) for consistency with CDC nomenclature.

VLY lyses target cells in a species-specific manner that is dependentupon the complement regulatory molecule CD59. In addition to causingerythrocyte lysis, VLY activates the conserved epithelial p38mitogen-activated protein kinase pathway and induces interleukin-8production by human epithelial cells. Transfection of human CD59 intonon-susceptible cells rendered them sensitive to VLY-mediated lysis. Inaddition, single amino acid substitutions in VLY (including some in theundecapeptide region such as (VLY(P480W)) generated toxoids that did notform pores. Introduction of the analogous proline residue into anotherCDC, pneumolysin, significantly decreased its cytolytic activity.

Certain embodiments of the present invention are directed to (1)isolated nucleic acid encoding VLY protein or a degenerate variant orfragment thereof from various strains of G. vaginalis, and to isolatedand purified VLY protein, or a biologically active fragment or variantthereof, (2) an isolated and purified nucleic acid encoding domain 4 ora degenerate variant or fragment thereof from various strains of G.vaginalis, and to isolated and purified domain 4 region of VLY, or abiologically active fragment or variant thereof, and (3) an isolated andpurified nucleic acid encoding the undecapeptide or a degenerate variantor fragment thereof from various strains of G. vaginalis, and theisolated and purified undecapeptide region of VLY, or a biologicallyactive fragment or variant thereof. So far we have found that theundecapeptide region is conserved.

Certain other embodiments are directed methods for diagnosing G.vaginalis infection by detecting the presence of VLY protein in abiological sample, preferably using a new ELISA with detectionantibodies to VLY.

Certain other embodiments are directed to various newly discoveredtoxoid forms of human VLY protein or an immunogenic fragment or variantthereof that has reduced pore-forming activity compared to wild-typeVLY. Another embodiment is directed to a composition comprising one ormore purified VLY toxoid proteins, and to a method of eliciting animmune response in an animal by introducing this toxoid composition intothe animal. Other compositions of the present invention include avaccine comprising one or more VLY toxoids.

Other embodiments include a method for permeabilizing a cell membrane(such as a cancer cell) to kill the cell or (in the case of cancer)render it more accessible to chemotherapeutic agents, comprisingcontacting the cell with a composition that includes purified VLYprotein or a biologically active fragment or variant thereof. Anotherembodiment is directed to VLY toxin that is bound to a molecule that istargeted specifically to the cancer cell or VLY toxin that is bound to achemotherapeutic agent.

We have discovered that VLY binds to CD59 receptor on epithelial cells,which enables it to permeabilize the vaginal epithelial cell. Thereforecertain embodiments are directed to methods for treating or preventingG. vaginalis and bacterial vaginosis by administering one or moreprotective agents (including soluble CD59 itself, and/or Anti CD59monoclonal or polyclonal antibodies, small molecules or anti-VLY andanti-PLY antibodies), that prevent VLY from binding to its CD59 receptoron target cells including vaginal epithelial confirm cells or otherwiseblock its pore-forming ability.

Using purified VLY toxin as an immunogen, we generated polyclonal rabbitimmune serum (IS) having a polyclonal rabbit anti-VLY antibody that wenamed “polyAnti-VLY.” PolyAnti-VLY inhibited VLY-mediated lysis of humancervical carcinoma cells and vaginal epithelial cells thereforepolyAnti-VLY has therapeutic use and can be used in an ELISA kit fordetecting VLY.

Certain embodiments of the invention are directed to methods that treatBV in order to reduce transmission of HIV from an HIV/BV-infected womanto her sexual partner or to a fetus she may be carrying, or to an infantduring childbirth by administering one or more of the describedprotective agents.

The G. vaginalis Genome Contains an Orthologue of Known CDCs

The CDC family is made up of more than 15 protein toxins produced byseveral distinct gram-positive genera (reviewed in (38)). The basiclocal alignment search tool (BLAST) was used to compare raw genomic DNAsequence data from the Gardnerella vaginalis 14018 genome project(available atttp://med.stanford.edu/sgtc/research/gardnerella_vaginalis.html(available at) with a database of known microbial genes (ComprehensiveMicrobial Resource, J. Craig Venter Institute). We identified a 1551base pairs (bp) open reading frame with 54% DNA sequence identity topneumolysin, the S. pneumoniae CDC that we proceeded to analyze. Thisgenomic region was amplified from G. vaginalis 14018 (ATCC) by PCR,cloned, and sequenced. See Example 1. SEQ ID NO: 1 sets forth the DNAsequence encoding VLY protein from G. vaginalis species 14018 and 14019,which are identical. VLY made by G. vaginalis strain 49145 is set forthin SEQ ID NO. 3. Certain embodiments are directed to nucleic acidsidentified by SEQ ID NO: 1 and 3, or a biologically active fragment orvariant thereof. Other embodiments are directed to a vector thatincludes the DNA SEQ ID NO: 1 or 3 encoding VLY protein from strains14018 and 14019, or 49145, respectively, or a biologically activefragment or variant thereof.

The predicted amino acid sequence of VLY (from strains 14018 and 14019)set forth in SEQ ID NO. 2, exhibits sequence similarity and identityconsistent with reported relationships among members of the CDC family(Appendix). The predicted sequence of VLY from G. vaginalis strain (ATCC49145) is identical with the exception of a single amino acidsubstitution (R494H-See SEQ ID NO. 3). Certain embodiments are directedto the isolated and purified (naturally occurring and recombinant) VLYproteins described herein and to biologically active variants andfragments thereof, including those having the amino acid sequences setforth in SEQ ID NO: 2 for VLY from strains 14018-19), and the amino acidsequence set forth in SEQ ID NO: 11 for VLY from ARG3. An embodiment ofthe invention is directed also to Codon optimized VLY gene sequence(that has the same protein sequence as VLY from strain 14018) DNA SEQ IDNO: 9 which greatly improves yield and purity of recombinant toxin madein E. coli.

The phylogram of CDC protein sequences (FIG. 1A) demonstrates threedistinct groupings—a Streptococcus group (into which VLY falls), aListeria group, and a Bacillus/Clostridium group (also containing SLO).The members of the Streptococcus clade have the most divergence in thedomain 4 undecapeptide, including the presence of a proline residue asan insertion (pyolysin) or substitution (intermedilysin, VLY). In thecase of pyolysin, the unusual undecapeptide has been shown to berequired for pore formation (3). Seeligeriolysin, the CDC from L.seeligeri, has an alanine to phenylalanine substitution in theundecapeptide that causes a decrease in toxin efficacy compared tolisteriolysin 0 (21).

A phylogram of representative full-length CDC sequences (FIG. 1B)obtained from publicly available databases (Appendix) was constructedusing the neighbor-joining algorithm. By this analysis, VLY appears tobe most closely related to ILY and it falls within a group consisting ofmost of the CDC from genus Streptococcus, including pneumolysin,mitilysin, and suilysin. Pyolysin, from Arcanobacterium pyogenes (2), isthe least similar member of this group. VLY is more distantly related toCDC from the Bacillus, Listeria, and Clostridium genera, as well asstreptolysin O from Streptococcus pyogenes, which is divergent from theother streptococcal CDC. Bootstrap analysis indicates a high degree ofconfidence for the placement of VLY in the streptococcal group (data notshown).

The undecapeptide is an 11 amino acid sequence in domain 4 of the CDCsthat is well conserved and of particular importance for host cellinteraction and pore formation (38). The nucleic acid sequences encodingthe undecapeptide of G. vaginalis are set forth in DNA SEQ ID NO: 6 forstrains 14018 and 14019 (nucleic acid residues 1414-1446), and DNA SEQID NO: 14 for strain 49145. The VLY undecapeptide is divergent from theCDC consensus sequence at 3 of 11 sites (FIG. 1B), one of which is analanine to valine substitution. More strikingly, there is a prolinesubstitution at VLY position 480, the site of one of the conservedtryptophan residues that is important for pore formation in other CDCfamily members (24). The loss of the conserved cysteine residue at thesecond position of the undecapeptide is seen in two other CDC, ILY andpyolysin, and is consistent with the prior report of insensitivity ofthe G. vaginalis toxin to reducing agents (35). Western blot analysis oflysed G. vaginalis bacteria demonstrates a ˜57 kD band that cross reactswith a monoclonal antibody directed against the S. pneumoniae CDC,pneumolysin.

Purified, recombinant human-specific VLY (from strains 14018 and 14019)identified by amino acid or a fragment or variant thereof, that alsocomes within the scope of this invention, migrates at a similarmolecular weight to natural VLY and is also detected by anti-pneumolysinantibody (FIG. 1C). One embodiment of the invention is directed totreating a G. vaginalis infection by administering anti-pneumolysinantibody and/or anti-VLY antibody, preferably locally in the form of agel vaginal suppository.

Our results show that domain 4 stretches from the conserved tyrosine atposition 376 (amino acid position) to the end of the protein which is516 amino acids long. Certain embodiments are directed to thecorresponding DNA SEQ ID NO: 4 encoding domain 4 that begins atnucleotide 1126 and continues to the end of the DNA molecule for strains14018-9 and SEQ ID NO: 12 (for strain 49145), and to amino acidsequences of domain 4 as set forth in SEQ ID NO: 5 (for strains 14018-9)and SEQ ID NO: 13 (for strain 49145).

Species-specific, Cholesterol-dependent Hemolytic Activity of VLY

Recombinant VLY produced in E. coli was used for studies of toxininteraction with target cells. Purified recombinant VLY lysed primaryhuman erythrocytes in a dose-dependent fashion (FIG. 2A). In contrast,sheep (FIG. 2A-B), mouse (FIG. 2B) and horse (data not shown)erythrocytes were resistant to lysis even at substantially higher VLYconcentrations. Erythrocytes from all of these species were lysed bypneumolysin, a non-species-restricted member of the CDC family (FIG.2B). Preincubation of VLY with cholesterol inhibited lysis of humanerythrocytes in a dose-dependent manner, consistent with itsclassification within the CDC family (FIG. 2C). Thus VLY is speciesspecific for lysing human cells.

We have also noted profound ultrastructural changes in epithelial cellsexposed to sublytic quantities of VLY (about 250 ng/ml) using live-cellimaging of HeLa cells. This response occurred rapidly (<1 min) followingexposure of cells to VLY, and blebs remained intact and present over thecourse of hours prior to resolution. These blebs were contiguous withthe epithelial cell cytoplasm, as demonstrated with hCD59-IRES-GFPtransfected CHO cells, in which cytoplasmic GFP was observed enteringthe blebs. Most strikingly, these ultrastructural changes wererecapitulated by antibody-mediated cross-linking of the hCD59 receptorand by binding of a non-pore-forming GFP-VLY-D4 fusion protein. Thesefindings are consistent with signaling through hCD59 as a potentialmechanism for initiating ultrastructural changes. We hypothesize thatthis bleb formation represents a novel pathway of toxin recognition byepithelial cells' unique response to hCD59-dependent toxins and involvessignaling pathways initiated by hCD59 binding.

VLY Lytic Activity Requires Binding to Complement Regulatory MoleculeCD59 on the Surface of Epithelial Cells

The human specificity of the G. vaginalis hemolysin was noted in earlierstudies (8), but no specific mechanism for this was described. Therecent characterization of human CD59 as a receptor for intermedilysin(ILY) represented a major step forward in the understanding of themechanism of action of CDC, which were previously thought to bindcholesterol directly as the sole requirement for pore-formation. Theresults presented below show that that this model is oversimplified andthat at least a subset of the CDCs including VLY require proteinreceptors on the surface of target cells (15).

The results in FIG. 3A show that blockade of CD59 on the surface ofprimary human erythrocytes using monoclonal antibody MEM-43 cloneabrogated VLY-induced lysis. By contrast, antibody against anothererythrocyte surface marker CD55 was ineffective, anti-CD59 antibodiesfor use in treating G. vaginalis and bacterial vaginosis infections arecommercially available for example from- Santa Cruz Biotechnology andGenetex. Thus surface CD59 on epithelial cells is the receptor for VLYand binding of CD59 by VLY is necessary for lytic activity.

Our results showed that the activity of pneumolysin, a CDC that does notexhibit host specificity, was not inhibited by either anti-CD59 oranti-CD55 (FIG. 3B). However, transfection of human CD59 into Chinesehamster ovary (CHO) cells significantly increased lactate dehydrogenaserelease in the setting of treatment with VLY (FIG. 3C) but notpneumolysin (FIG. 3D), indicating that CD59 is sufficient to confersusceptibility on at least a subset of VLY resistant cells.

CD59 is expressed on the surface of human genital tract epithelialcells, the target cell type during G. vaginalis colonization and BV, asdemonstrated by immunofluorescence staining with monoclonal antibodyagainst hCD59 (MEM-43 clone). Secondary anti-mouse IgG-Alexa Fluor 488(green) was used. hCD59 has also been shown to be highly expressed inthe female genital tract in vivo, along with other complement regulatorymolecules (70).

Certain embodiments of the invention are directed to methods fortreating G. vaginalis infection by (1) administering soluble CD59 itself(CD59 with the GPI anchor deleted) to saturate or neutralize VLY therebypreventing it from binding to CD59 receptor on vaginal epithelial cells,or (2) administering anti-CD59 or anti-VLY polyclonal or monoclonalantibodies that prevent binding of VLY to the CD59 receptor on vaginalepithelial cells. Monoclonal Anti-CD59 antibodies are commerciallyavailable from several sources.

Anti-VLY antibodies can be directed to the VLY protein or to abiologically active fragment or variant thereof, including domain 4 andthe undecapeptide regions of VLY. In a preferred embodiment, thetherapeutic anti-CD59 and anti-VLY antibodies and CD59 are locallyadministered, for example as a vaginal suppository. Such localadministration is not only efficient requiring fewer antibodies, but italso enables a high concentration of the therapeutic agent to bedelivered to the target cells. Since CD59 is ubiquitous and plays animportant role protecting cells from damage by complement, it is alsoimportant to specifically target CD59 expressed on the surface of thetarget epithelial cells, and not administer it systemically.

G. Vaginalis is a Gram-positive Bacterium

The discovery of the pore-forming toxin VLY in G. vaginalis expands theCDC family to another gram-positive genus and to a novel anatomic site.CDCs are produced by organisms that colonize and cause disease atmucosal surfaces including the upper and lower respiratory tracts andthe gastrointestinal tract. In many such cases, toxin production hasbeen shown to be essential for maintenance of colonization, pathogenesisof invasive disease, or both (38). CDCs have been described only ingram-positive organisms. Thus, the characterization of VLY and itsevolutionary relationship to the other CDC provides further evidencethat G. vaginalis is most properly grouped with the gram-positives,despite its variable staining characteristics (36). Antibiotics thathave been used to treat BV and G. vaginalis infections includemetronidazole, clindamycin and tinidazole.

Metronidazole is the most successful therapy. Most comparative studiesusing multiple divided-dose oral regimens for one week achieved earlyrates of clinical cure in excess of 90 percent, and cure rates (by Amselcriteria) of approximately 80 percent at four weeks. A randomized trialshowed that short-term cure rates were significantly higher when theinitial course of metronidazole therapy was 14 days rather than 7 days(Schwebke, J R, Desmond, R A. A randomized trial of the duration oftherapy with metronidazole plus or minus azithromycin for treatment ofsymptomatic bacterial vaginosis. Clin Infect Dis 2007; 44:213). However,long-term cure rates (21 days after completion of therapy) were similarfor both treatment regimens. In one embodiment, the oral regimen oftronidazole is 500 mg twice daily for seven days. Sexually TransmittedDiseases Treatment Guidelines, 2006. MMWR Recomm Rep 006. (RR-11);55:1-95. Topical vaginal therapy with 0.75 percent metronidazole gel (5g once daily for five days) is as effective as oral metronidazole. Thechoice of oral versus topical therapy depends upon patient preference.

Clindamycin can be used as a topical vaginal therapy with 2 percentclindamycin cream (5 g of cream containing 100 mg of clindamycinphosphate) as a seven-day regimen. Alternative regimens include oralclindamycin (300 mg twice daily for seven days) or clindamycin ovules(100 mg intravaginally once daily for three days) (Sexually TransmittedDiseases Treatment Guidelines, 2006. MMWR Recomm Rep 2006 (RR-11);55:1-95; Paavonen, J, Mangioni, C, Martin, M A, Wajszczuk, C P. Vaginalclindamycin and oral metronidazole for bacterial vaginosis: a randomizedtrial. Obstet Gynecol 2000; 96:256). A one-day or single application ofclindamycin as a bioadhesive has also been approved by the FDA(Clindesse). These regimens have not been studied extensively and mayhave lower efficacy for eradicating BV.

Tinidazole is a second generation nitroimidazole. It has a longerhalf-life than metronidazole (12 to 14 hours versus 6 to 7 hours) andfewer side effects (Tinidazole (Tindamax)—a new option for treatment ofbacterial vaginosis. Med Lett Drugs Ther 2007; 49:73). In oneembodiment, 1 g tindazole is administered orally once daily for fivedays, as efficacy is slightly higher and side effects are slightly lessfrequent than with shorter course therapy (tinidazole 2 g orally dailyfor two days).

VLY Toxoids: The Proline Residue in the Variant Undecapeptide of VLY(Residue 480) is Required for Cytolytic and Cell Stimulatory Activity

Although hemolysis is a useful model for toxin-induced pore formation,erythrocytes are unlikely to be a target cell for G. vaginalis undernormal physiologic conditions, as Gardnerella bacteremia is exceedinglyrare (11, 33). Activation of p38 mitogen-activated protein kinase (MAPK)is a conserved element in epithelial detection of bacterial pore-formingtoxins (32) and appears to be essential in defense of host cells fromtoxin attack (19). Exposure of the human cervical epithelial cell lineHeLa to VLY led to phosphorylation of p38 MAPK within 30 minutes (FIG.4A), consistent with epithelial responses to other pore-forming toxins(32). FIG. 4B shows that IL-8 mRNA is upregulated in HeLa cells by VLYand is dependent on there being a proline at position 480 in theundecapeptide. The data showing that VLY activates the p38 MAPK and IL-8pathways in human epithelial cells, shows that VLY produced by G.vaginalis as a major factor in the immunopathology of BV.

Nucleic acid and amino acid sequence alignments are set forth in theAppendix. At the protein level, the undecapeptide is identical G.vaginalis strains 14018 and 14019. DNA sequences encoding VLY areidentical for strains 14018 and 14019, and strains 49145 and ARG3 arevery close with several single base substitutions (seen in thealignment). For example, there is a single (silent) base pairsubstitution (G>A at nucleotide position 1428) in 49145 compared to theother two (14018 and 14019). One nucleotide substitution leads to achange in VLY protein sequence (R->H at amino acid position 494) in the49145 strain. This change is in domain 4 but after the undecapeptideregion.

Genetic and structural studies have implicated domain 4 (D4) of the CDCas being crucial for membrane association. Specifically, theundecapeptide appears to have a significant role in CDC function.Because of the importance of the undecapeptide to toxin function, wecreated a mutant named VLY(P480W) using site-directed mutagenesis tomake a single amino acid substitution of tryptophan was for proline atposition 480. This change converted the proline residue in the VLYundecapeptide to the consensus tryptophan residue, which has been shownto be crucial to the function of several other CDC that contain theconsensus undecapeptide.

Notably, the P480W mutation produced a VLY toxoid that was substantiallyless effective at lysing human erythrocytes (FIG. 4C). In addition, thisproline to tryptophan mutation abolished p38 activation and IL-8transcription in HeLa cells (FIG. 4A-B) compared to wild-type VLY (FIG.4A-B). This proline residue does not dictate species-specificity butappears to be crucial for toxin function. These findings underscore theimportance of D4, and specifically the undecapeptide, to CDC function,even among the hCD59-dependent CDCs. These results confirm that prolineat amino acid residue 480 of VLY is necessary for efficientpore-formation and cell activation by VLY. Because the P480W toxoid issignificantly less toxic (lytic activity is reduced) than native VLYtoxin, it can be used in vaccines that elicit a specific immune responseto VLY toxin.

In order to investigate further the potential role of proline in poreforming activity, we made the converse mutation at the correspondinglocation (W435P) in pneumolysin, the species-non-specific CDC from S.pneumoniae. This pneumolysin mutant lysed erythrocytes, but only atconcentrations much higher than wild-type toxin (FIG. 4D). Thisindicates that a substitution of tryptophan (W) for proline caused aloss of pore-forming activity. Construction of the correspondingmutation in PLY (i.e. substitution of proline for tryptophan at position435) similarly led to a substantial decrease in its lytic activity.

Certain embodiments of the invention are also directed to toxoids of PLYand penumolysin, wherein there is an amino acid substitution oftryptophan for proline in the undecapeptide, and to the use of the PLYtoxoids to elicit an immune response in a host.

These findings emphasize the importance of the structure of theundecapeptide region to the function of CDCs. Likewise, the substitutionof a lysine residue for the conserved cysteine in the undecapeptide is amodification unique to VLY. Prior reports have demonstrated that the G.vaginalis hemolysin is not thiol-activated (35). The lack of enhancingeffect of a reducing agent is consistent with this modification in theundecapeptide. Of note, in other CDC family members, the conservedcysteine residue confers thiol-activating properties but is notessential for pore-forming activity (37).

The following newly identified toxoids all have decreased pore-formingactivity compared to the wild-type toxin, and are therefore useful inmaking a vaccine or antibodies against VLY or eliciting an immuneresponse in a patient against G. vaginalis. Certain embodiments aredirected to the toxoids listed below:

P480W, described above

N500I at position 500 change asparagines to isoleucine.

P480W, N500I double mutant

V471R, K473C double mutant

V471R, K473C, P480W triple mutant

Other embodiments are directed to toxoids in which:

1. Some or all of domain 4 is deleted. (a portion of the sequence can beremoved, or substituted or a premature stop codon can be inserted); and

2. Some or all of the amino acids in the undecapeptide (aa472-483) arereplaced with alanine or phenylalanine; substitutions for the proline atposition 480 in native VLY as stated before are particularly useful.

Certain embodiments of the present invention are directed to isolatedand purified VLY recombinant toxoid proteins or an immunologic fragmentor variant thereof, including P480W, N500I, V471R, K473C, P480W-N500Idouble mutant, V471R-K473C double mutant, and V471R-K473C-P480W triplemutant, or immunologic fragments or variants thereof, and to otherpurified toxoids described herein (including purified recombinantforms). Other embodiments include recombinant purified VLY protein ortoxoids that have a 6×His tag on both the N-terminus (beginning) andC-terminus (end) of the protein to maximize purification. The tags canbe removed after the VLY protein or toxoids are isolated and purified.

Other embodiments are directed to a composition comprising any of thesetoxoids or an immunologic fragment or variant thereof, for example to beused as immunogens in vaccines. Other embodiments are directed to amethod for generating an immunologic response to VLY in an animal byadministering to a patient a composition that includes one or more VLYtoxoids such as the VLY (P480W) toxoid (or an immunologic fragment orvariant thereof.

New Rabbit Polyclonal Anti-VLY Antibodies Detect G. vaginalis by WesternBlot and Immunofluorescence

Recombinant purified VLY toxin generated and purified as described inthe Examples was submitted to Cocalico Biologicals, Inc. (Reamstown,Pa.). According to their protocol, adult rabbits were injected with aminimum of 100 μg antigen mixed with Complete Freund's Adjuvantsubcutaneous and/or intramuscularly at multiple sites. Booster dosescontaining a minimum of 50 μg antigen mixed with Incomplete Freund'sAdjuvant were administered on days 14, 21 and 49. A test bleed wasperformed on day 56. Prior to the first immunization, serum wascollected from each rabbit to serve as negative control. We named thenew anti-VLY polyclonal antibody thus derived “polyAnti-VLY”; certainembodiments of the invention are directed to this antibody and itstherapeutic use to treat G. vaginalis infections and BV, and to preventtransmission of HIV to another from a woman infected with G. vaginalisor having BV.

Western blot analysis of lysed G. vaginalis 14018 revealed a single bandusing polyclonal immune serum (polyAnti-VLY) as the primary antibody(FIG. 5A). This corresponds to the predicted 57 kDa molecular mass ofVLY and to our prior findings using cross-reacting anti-pneumolysinantibody (50). There were no visible bands detected on membranes probedwith pre-immune serum and processed identically (data not shown).Immunofluorescent detection of VLY associated with whole G. vaginaliswas detected microscopically using immune serum and fluorescentlylabeled anti-rabbit secondary antibodies (FIG. 5B). Preimmune serum didnot lead to detectable fluorescence of G. vaginalis (FIG. 5B).

Certain embodiments of the present invention are directed to the newrabbit polyAnti-VLY antibody, and fragments and variants thereof and tocompositions that include the antibody that are intended for use in amammal, particularly a human. Other embodiments are directed generallyto polyclonal antibodies to VLY made by immunizing an animal with theisolated recombinant VLY toxins or a VLY toxoid as herein described, oran immunologically active fragment or variant thereof. Anti-VLYantibodies will bind to free VLY in the body. Other embodiments aredirected to monoclonal anti-VLY antibodies made using well knownhybridoma technology. In certain embodiments the anti-VLY antibodies canbe humanized (as antibody or antibody fragments) and then administeredtherapeutically to a patient to confer passive immunity to G. vaginalisor BV or to neutralize VLY either systemically or locally in the vaginato prevent it from binding to CD 59. The antibodies can also be used inELISA or RIA assays to detect the presence of VLY in a biological samplefrom a patient as is described below.

New ELISA that Detects VLY Production by G. vaginalis

We developed a sandwich ELISA assay capable of quantifying VLY at ng/mlconcentrations in the supernatant of growing G. vaginalis using rabbitpolyAnti-VLY (diluted 1:1000 in blocking solution) as the detectionantibody and known concentrations of recombinant VLY toxin diluted in G.vaginalis culture medium as a standard. As a secondary antibody we usedgoat anti-rabbit HRP antibody 1:100 dilution). We found that VLY toxinproduction peaked at between 24 and 36 hours of G. vaginalis in culture(FIG. 6A) and directly correlated with bacterial concentration asdetermined by optical density (FIG. 6 B). The ELISA technique is robust,even in the setting of potential inhibitors (such as serum) and will beuseful for quantifying VLY production both in vitro and in vivo. TheELISA based assay in particular, is sensitive, robust and directlycorrelates with the concentration of G. vaginalis, reported to be anindependent predictor of BV and subsequent preterm delivery. (61-65)

As an alternative means to assess VLY regulation, we also developed asensitive real-time PCR assay targeting the VLY gene. This assay, whichcan be used either to detect DNA from organisms with the toxin gene orto monitor VLY RNA levels during different G. vaginalis growthconditions, represents an important strategy for our continuedinvestigations.

Certain embodiments of the invention are directed to a kit for detectingVLY toxin or fragment that includes an anti-VLY antibody (includingrabbit polyAnti-VLY) as the detection antibody, or anti-pneumolysinantibody, anti-PLY antibody, anti-ILY antibody or any other antibodythat cross reacts with VLY. Such a kit can be used as a diagnostic toolfor G. vaginalis infections and bacterial vaginosis by testing thepresence of VLY in a biological sample from a patient. The biologicalsample is preferably a vaginal swab that has been diluted in sterilesaline or PBS before the assay is run. It is expected that the swab willhave not only vaginal cells, but also free VLY toxin. Another sample canbe obtained by instilling 1-5 ml sterile saline into the vaginal ofpatient, and then collecting a biological sample from the vagina for theassay.

An embodiment of a kit for a sandwich ELISA optionally includes asecondary antibody (specific for the detection antibody) conjugated toan enzyme or other compound known in the art (including fluorescentlabels/biotin avidin, radiolabels) that permits detection of the bindingof the secondary antibody to the detection antibody.

In one embodiment the rabbit polyAnti-VLY is conjugated to an enzyme foran indirect assay. A major disadvantage of the indirect ELISA is thatthe method of antigen immobilization is non-specific; any proteins inthe sample will stick to the microtiter plate well, so smallconcentrations of analyte in serum must compete with other serumproteins when binding to the well surface. The sandwich ELISA provides asolution to this problem: (1) Plate is coated with a capture antibody;(2) sample is added, and any antigen present binds to capture antibody;(3) detecting antibody is added, and binds to antigen; (4) enzyme-linkedsecondary antibody is added, and binds to detecting antibody; (5)substrate is added, and is converted by enzyme to detectable form.

In yet another embodiment the kit includes a capture antibody that bindsVLY toxin to the substrate, thereby preventing nonspecific binding ofpeptides or proteins in the sample. The capture antibody can beanti-penumolysin antibody that we used (such as clone 1F11 or otherclones that cross-reacts with VLY), or soluble CD59, anti-VLY antibodiesincluding polyAnti-VLY, anti-PLY antibody, anti-pneumolysin, ormonoclonal anti-VLY.

An ELISA may be run in a qualitative or quantitative format. Qualitativeresults provide a simple positive or negative result for a sample. Thecutoff between positive and negative is determined by the analyst andmay be statistical. Two or three times the standard deviation is oftenused to distinguish positive and negative samples. In quantitativeELISA, the optical density or fluorescent units of the sample isinterpolated into a standard curve, which is typically a serial dilutionof the target.

Antiserum Against VLY Inhibits Toxin-mediated Cytolysis

To test the biological activity of our antiserum to VLY, we studiedtoxin-mediated cytolysis of human erythrocytes, which are susceptible tohemolysis at various concentrations of purified recombinant VLY for 30minutes (FIG. 7A). Pre-incubation of VLY with polyAnti-VLY (immune serum1:50 dilution) prior to exposure to human erythrocytes resulted insignificantly less hemolysis compared to untreated cells or cellsexposed to pre-immune serum-treated VLY (FIG. 7A) Inhibition ofVLY-mediated lysis by immune serum was dose-dependent (FIG. 7B).

Similarly, VLY-mediated cell lysis of human epithelial cell lines HeLa(FIG. 8A) and VK2 (FIG. 8B) was markedly reduced in the setting ofimmune serum. In order to generate a probe for toxin-hCD59 interactions,we created a GFP:VLYD4 fusion protein (not shown). This protein binds tohCD59-expressing human epithelial cells but does not form pores. It alsocan be used therapeutically to treat BV or G. vaginalis infections.

Treatment of G. vaginalis Infections and BV

The results above show that anti-VLY antibodies in general andpolyAnti-VLY in particular can be used therapeutically to treat G.vaginalis infections by neutralizing free VLY. Such antibodies can beadministered locally in the vagina as a topical formulation, orsystemically. When the anti-VLY antibodies are administeredlocally/topically to the vagina, It is expected that the disadvantagesof using rabbit antibodies instead of human antibodies is minimized.

New compositions that come within the scope of the invention fortreating BV or G. vaginalis infections, or for preventing transmissionof HIV from a BV/HIV-infected woman include:

-   -   polyclonal and monoclonal anti-VLY antibodies (hereafter        “anti-VLY antibodies”) including the new anti-VLY antibody we        described and named “rabbit polyAnti-VLY”

Compositions comprising two or more of the following:

-   -   anti-VLY antibodies, or fragments or variants thereof    -   soluble CD59 or a fragment or variant thereof that binds to VLY    -   anti-CD59 monoclonal or polyclonal antibodies that prevent VLY        from binding to CD59 on the surface of epithelial cells or that        prevent activation of surface CD59 by VLY, or fragments or        variants thereof    -   antibiotics known to be used in the treatment of BV or G.        vaginalis infections, described herein    -   anti-pneumolysin antibody, or fragments or variants thereof    -   anti-VLY antibody, or fragments or variants thereof    -   VLY toxoids which will compete with VLY toxin for binding to        surface CD59 and that can be used for vaccines.        Reducing the Transmission of HIV by an Infected Woman

BV has been repeatedly associated with both a significant risk of HIVacquisition and increased viral shedding among those already infected43-45. In vitro, treatment of HIV-infected cells with Gardnerella leadsto increased production of viral transcripts. J Infect Dis 179: 924,Lancet 353: 525, and J Bacteriol 190: 3896, incorporated herein byreference. Treatment of HIV-infected U-1 cells with purified VLY toxinin vitro caused a significant increase in HIV transcripts and p24release (Unpublished observations). Therefore, treatment or preventionof BV in women will also greatly reduce their susceptibility to becominginfected with HIV. An embodiment of the invention is directed to methodsfor reducing or preventing the transmission of HIV by a woman infectedwith both HIV and BV to another through intercourse by administering tothe woman before she engages in a sexual activity, a therapeuticallyeffective amount of a composition comprising a protective agent asdescribed herein. In one embodiment the agent is applied topically tothe vagina of the infected woman before engaging in the sexual activity.Vagina and birth canal are used synonymously herein.

The woman can be treated either topically or systemically or both. Theprotective agent(s) bind to VLY, or bind to CD59 receptor on targetcells blocking the binding of VLY. In one embodiment, VLY toxoids asdescribed herein bind to CD59 thereby preventing the toxin VLY frombinding and lysing cells. Without being bound by theory we speculatethat the protective agent neutralizes VLY produced from G. vaginalisinfected cells thereby preventing it from binding to CD59 receptor onHIV-infected cells; this prevents VLY from increasing viral shedding byHIV-infected cells, which in turn reduces the risk of HIV infection tothe uninfected sexual partner. A therapeutically effective amount of theprotective agent is an amount that reduces or prevents transmission ofHIV, preferably by reducing shedding by HIV-infected cells therebyreducing the viral load of HIV in a biological sample taken from thebirth canal of the woman. A therapeutically effective amount thatreduces viral load in a biological sample can be determined usingroutine experimentation.

Approximately 7,000 human immunodeficiency virus (HIV)-infected womengive birth in the United States each year. This number is many timeshigher in Africa. Without treatment, about one-fourth of them transmitthe virus to their children. The anti-HIV drug zidovudine (AZT), givento HIV-infected pregnant women before and during childbirth and to theirinfants after childbirth, reduces HIV transmission by as much astwo-thirds. Treatment with AZT is now the standard of care in the U.S.for preventing HIV infection in infants. However, additional means areneeded for the prevention of maternal to fetal transmission of HIV andother enveloped viruses both in the U.S. and worldwide. We havediscovered compositions and methods that reduce the risk of a woman whois both HIV and BV positive transmitting HIV to an unborn fetus duringthe birth process.

One embodiment of the invention is directed to a method of reducingmaternal to fetal transmission of HIV where the pregnant woman isinfected with both HIV and BV, by administering a therapeuticallyeffective amount of a protective agent either topically or systemically,or both, especially before and during a vaginal birth. Treatment of thepregnant HIV- and BV-infected individual should begin as soon as she isidentified to reduce the viral load in the peripheral blood of the womanto both treat the BV and reduce the risk of the fetus becoming infectedwith HIV either before or during the birth.

When birth is approaching, treatment should be undertaken during laboruntil birth, with repeated topical application. Routine experimentationwill determine the optimum schedule of administration. A therapeuticallyeffective amount includes an amount that reduces or eliminates free HIVvirus in a biological sample from the pregnant woman, such as a bloodsample or a sample of vaginal secretion.

In another embodiment for preventing transmission of BV, G. vaginalis orHIV to a newborn, the preventative agent is administered topically tothe newborn immediately after birth, for example, to an exposed tissueof the newborn such as the umbilical cord. The application of thepreventive agent directly to the newborn can contribute to the reductionor elimination of HIV viral particles that may be shed by anyHIV-infected cells that remain from maternal-derived biological materialon the newborn.

The methods described herein to reduce or prevent maternal to fetaltransmission of an enveloped virus can be used in combination with oneor more known treatments of HIV or BV, including using antiviral agentsor antibacterial agents.

Antibodies for Use in the Present Invention

“Antibody” or “antibodies” as described herein include intact moleculesas well as fragments thereof that are capable of binding to an epitopeof a VLY polypeptide or VLY toxoid, as described herein, to a fragmentor variant thereof. The term “epitope” refers to an antigenicdeterminant on an antigen to which an antibody binds. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains, and typically have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. Epitopes generally have at least five contiguous aminoacids. The terms “antibody” and “antibodies” include polyclonalantibodies, monoclonal antibodies, humanized or chimeric antibodies,single chain Fv antibody fragments, Fab fragments, and F(ab)₂ fragments.Polyclonal antibodies are heterogeneous populations of antibodymolecules that are specific for a particular antigen, while monoclonalantibodies are homogeneous populations of antibodies to a particularepitope contained within an antigen. Monoclonal antibodies areparticularly useful.

The term “specifically binds” as used herein refers to the situation inwhich one member of a specific binding pair does not significantly bindto molecules other than its specific binding partner(s) as measured by atechnique available in the art, e.g., competition ELISA. The term isalso applicable where e.g. an antigen-binding domain of an antibody ofthe invention is specific for a particular epitope that is carried by anumber of antigens (for example by VLY or fragment thereof such asdomain 4 and the undecapeptide, or a VLY toxoid), in which case theantibody carrying the antigen-binding domain will be able tospecifically bind to the various antigens carrying the epitope. The term“epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody at one or more of the antibody'santigen-binding regions.

“Specific binding” of an antibody also means that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷ M⁻¹, preferably greaterthan 10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities.

Antibody fragments that have specific binding affinity (as definedbelow) for VLY polypeptides can be generated by known techniques. Suchantibody fragments include, but are not limited to, F(ab′)₂ fragmentsthat can be produced by pepsin digestion of an antibody molecule, andFab fragments that can be generated by deducing the disulfide bridges ofF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed. See, for example, Huse et al. (1989) Science 246:1275-1281.Single chain Fv antibody fragments are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge (e.g.,15 to 18 amino acids), resulting in a single chain polypeptide. Singlechain Fv antibody fragments can be produced through standard techniques,such as those disclosed in U.S. Pat. No. 4,946,778.

Once produced, antibodies or fragments thereof can be tested forrecognition of a VLY polypeptide or toxoid by standard immunoassaymethods including, for example, enzyme-linked immunosorbent assay(ELISA) or radioimmunoassay assay (RIA). See, Short Protocols inMolecular Biology eds. Ausubel et al., Green Publishing Associates andJohn Wiley & Sons (1992). Suitable antibodies typically have equalbinding affinities for recombinant and native proteins.

An “antibody” refers to an intact immunoglobulin or to anantigen-binding portion thereof that competes with the intact antibodyfor specific binding. Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, inter alia, Fab,Fab′, F(ab′).sub.2, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide. An “immunoglobulin” is a tetrameric molecule. In anaturally-occurring immunoglobulin, each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy terminal portion of each chain defines aconstant region primarily responsible for effector function.

The monoclonal antibodies for use in the invention may be obtained byany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique of Kohler and Milstein, (1975, Nature256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. NatL Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclasses thereof. The hybridoma producing the mAb may be cultivated invitro or in vivo.

Human light chains are classified as kappa and lambda light chains.Heavy chains are classified as mu, DELTA, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairform the antibody binding site such that an intact immunoglobulin hastwo binding sites. Immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. From Nterminus to C terminus, both light and heavy chains comprise the domainsFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acidsto each domain is in accordance 44 with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901 917 (1987); Chothia et al. Nature 342:878 883 (1989).

An Fab fragment is a monovalent fragment consisting of the VL, VH, CLand CH I domains; a F(ab′).sub.2 fragment is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a Fd fragment consists of the VH and CHI domains; an Fv fragmentconsists of the VL and VH domains of a single arm of an antibody; and adAb fragment (Ward et al., Nature 341:544 546, 1989) consists of a VHdomain. A single-chain antibody (scFv) is an antibody in which a VL andVH regions are paired to form a monovalent molecules via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423 426, 1988 and Huston et al., Proc. Natl. Acad. Sci.USA 85:58795883, 1988). Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites. (See, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA90:6444 6448, 1993, and Poljak, R. J., et al., Structure 2: 1121 1123,1994.) One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an immunoadhesin. Animmunoadhesin may incorporate the CDR(s) as part of a larger polypeptidechain, may covalently link the CDR(s) to another polypeptide chain, ormay incorporate the CDR(s) noncovalently. The CDRs permit theimmunoadhesin to specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody hastwo different binding sites. An “isolated antibody” is an antibody that(1) is not associated with naturally associated components, includingother naturally-associated antibodies, that accompany it in its nativestate, (2) is free of other proteins from the same species, (3) isexpressed by a cell from a different species, or (4) does not occur innature.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In a preferred embodiment, all of the variable and constantdomains are derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways, asdescribed below.

A humanized antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Alternatively, a humanizedantibody may be produced by fusing the constant domains from a humanantibody to the variable domains of a non-human species. Examples of howto make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293, incorporated herein by reference.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. Fragments or analogs of antibodies can be readilyprepared by those of ordinary skill in the art following the teachingsof this specification. Preferred amino- and carboxy-termini of fragmentsor analogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991).

Pharmaceutical Compositions

The present invention also includes pharmaceutical compositions andformulations of the protective agents described herein. Pharmaceuticalcompositions of the present invention contain the therapeutic agent inan amount sufficient to prevent or treat the diseases described hereinin a subject. These pharmaceutical compositions are suitable foradministration to a subject in need of prophylaxis or therapy for any ofthe described diseases or conditions. The subject is preferably a humanbut can be non-human as well. A suitable subject can be an individualwho is suspected of having, has been diagnosed as having, or is at riskof developing one of the described diseases.

In some embodiments the protective agent is formulated in a lubricant asdescribed in Porat, U.S. Pat. No. 624,198, incorporated herein byreference, for intra-vaginal application or application to a newbornbaby. In a preferred embodiment the lubricant-protective agentcomposition has a natural pH corresponding to that of the vagina. Thelubricant may be any effective lubricant or combination of lubricantsacceptable for cosmetic applications. Medical and pharmaceutical studieshave shown that HIV develops mainly in the blood cells and is carried byvarious body fluids to other cells. The lubricant reduces the frictionbetween the penis and the vaginal wall, thus reducing the rupture ofblood cells which might otherwise occur and therefore reducing theamount of blood that is commingled. The protective agents of the presentinvention can also be formulated into gels and foams for applicationbefore or during sexual intercourse that are known in the art. In someembodiments the protective anti-shedding agents of the invention areincluded in disinfectant foam that coats the walls of the vagina andtemporarily forms a closed layer of foam that traps and kills any HIVvirus that may be present in addition to reducing or preventing furthershedding. Schmittmann, et al., U.S. Pat. No. 6,022,545, incorporatedherein by reference.

The preventive agent can be formulated into any topical compositionknown in the art that is suitable for its intended use as describedherein, including creams, lotions, ointments, gels, lubricants, liquids,sprays, powders, or absorbent materials. The preventative agents canalso be formulated for systemic administration for treating orpreventing G. vaginalis or BV, or for use as a vaccine, or for reducingshedding in HIV-BV infected women as described herein. A composition ofthe preventive agents for either topical or systemic administration canalso include a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antiviral agents, antibacterial agents,antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions. Othertopical formulations for preventing or reducing HIV transmission duringbirth are described in Sheele et al., U.S. Pat. No. 7,151,091, which isincorporated herein by reference.

Therapeutic compositions may contain, for example, such normallyemployed additives as binders, fillers, carriers, preservatives,stabilizing agents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70% active ingredient.

The protective agents can also be mixed with diluents or excipientswhich are compatible and physiologically tolerable. Suitable diluentsand excipients are, for example, water, saline, dextrose, glycerol, orthe like, and combinations thereof. In addition, if desired, thecompositions may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, stabilizing or pH buffering agents.

In some embodiments, the therapeutic compositions of the presentinvention are prepared either as liquid solutions or suspensions, assprays, or in solid forms. Oral formulations usually include suchnormally employed additives such as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%. One example of an oral composition useful fordelivering the therapeutic compositions of the present invention isdescribed in U.S. Pat. No. 5,643,602 (incorporated herein by reference).

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, pessary, transdermal patches, ointments,gels, lubricants, liquid, sprays, powders, absorbent materials, andsuppositories. For salves and creams, traditional binders, carriers andexcipients may include, for example, polyalkylene glycols ortriglycerides. One example of a topical delivery method is described inU.S. Pat. No. 5,834,016 (incorporated herein by reference). Otherliposomal delivery methods may also be employed (See, e.g., U.S. Pat.Nos. 5,851,548 and 5,711,964, both of which are herein incorporated byreference). The composition can include an inert carrier. Thecomposition can be impregnated in a towlette, sponge or capsule.

The formulations may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

Sustained-release preparations may also be prepared. Suitable examplesof sustained release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the protective agents, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained release matrices include, but arenot limited to, polyesters, hydro gels (for example, poly(2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides,copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT (injectable micro spheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

The protective agents of the present invention may be administered byany suitable means, preferably topically in the birth canal but alsoincluding systemic routes, such as parenteral, and subcutaneous.Parenteral infusions include intramuscular, intravenous, intra-arterial,intra-peritoneal, or subcutaneous administration.

For the prevention or treatment of disease, the appropriate dosage ofantibody or other protective agent will depend on the type of disease tobe treated, the severity and course of the disease, whether the drug isadministered for protective or therapeutic purposes, previous therapy,the patient's clinical history and response to the drugs and thediscretion of the attending physician.

The protective agents and vaccines are suitably administered to thepatient at one time or over a series of treatments.

The amount of antibody to be administered therapeutically rangestypically from about 1 ug to 100 ug/ml. This amount typically varies anddepends on the route of administration. The therapeutic agents of theinvention can be administered by one or more separate administrations,topical or systemic administration, or by continuous infusion. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until the symptoms of G. vaginalisor BV are sufficiently reduced or eliminated, or until the HIV viralload is reduced in women with HIV and BV. The progress of this therapyis easily monitored by conventional techniques and assays, and may beused to adjust dosage to achieve a therapeutic effect.

Protein Modifications

VLY protein and toxoids, and their biologically active analogs,derivatives, fragments and variants for use in the present invention canbe modified according to known methods in medicinal chemistry toincrease its stability, half-life, uptake or efficacy. Certain knownmodifications are described below.

As is also well known, polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination,and they may be circular, with or without branching, generally as aresult of post-translation events, including natural processing eventsand events brought about by human manipulation which do not occurnaturally. Circular, branched and branched circular polypeptides may besynthesized by non-translational natural processes and by syntheticmethods.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.Blockage of the amino or carboxyl group in a polypeptide, or both, by acovalent modification, is common in naturally-occurring and syntheticpolypeptides. For instance, the amino terminal residue of polypeptidesmade in E. coli, prior to proteolytic processing, almost invariably willbe N-formylmethionine.

The modifications can be a function of how the protein is made. Forrecombinant polypeptides, for example, the modifications will bedetermined by the host cell posttranslational modification capacity andthe modification signals in the polypeptide amino acid sequence.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells, and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications. The same type of modification may be present in the sameor varying degree at several sites in a given polypeptide. Also, a givenpolypeptide may contain more than one type of modification.

VLY protein and toxoids can be isolated and purified from cells thatnaturally express it, purified from cells that naturally express it buthave been modified to overproduce it, e.g., purified from cells thathave been altered to express it (recombinantly), synthesized using knownprotein synthesis methods, or by modifying cells that naturally encodeVLY or VLY toxoid to express it.

Protein Modification Description Acetylation Acetylation of N-terminusor e-lysines. Introducing an acetyl group into a protein, specifically,the substitution of an acetyl group for an active hydrogen atom. Areaction involving the replacement of the hydrogen atom of a hydroxylgroup with an acetyl group (CH₃CO) yields a specific ester, the acetate.Acetic anhydride is commonly used as an acetylating agent, which reactswith free hydroxyl groups. Acylation may facilitate addition of otherfunctional groups. A common reaction is acylation of e.g., conservedlysine residues with a biotin appendage. ADP-ribosylation Covalentlylinking proteins or other compounds via an arginine- specific reaction.Alkylation Alkylation is the transfer of an alkyl group from onemolecule to another. The alkyl group may be transferred as an alkylcarbocation, a free radical or a carbanion (or their equivalents).Alkylation is accomplished by using certain functional groups such asalkyl electrophiles, alkyl nucleophiles or sometimes alkyl radicals orcarbene acceptors. A common example is methylation (usually at a lysineor arginine residue). Amidation Reductive animation of the N-terminus.Methods for amidation of insulin are described in U.S. Pat. No.4,489,159. Carbamylation Nigen et al. describes a method ofcarbamylating hemoglobin. Carboxylation Carboxylation typically occursat the glutamate residues of a protein, which may be catalyzed by acarboxylase enzyme (in the presence of Vitamin K - a cofactor).Citrullination Citrullination involves the addition of citrulline aminoacids to the arginine residues of a protein, which is catalyzed bypeptidylarginine deaminase enzymes (PADs). This generally converts apositively charged arginine into a neutral citrulline residue, which mayaffect the hydrophobicity of the protein (and can lead to unfolding).Condensation of amines Such reactions, may be used, e.g., to attach apeptide to other with aspartate or glutamate proteins labels. Covalentattachment of Flavin mononucleotide (FAD) may be covalently attached toflavin serine and/or threonine residues. May be used, e.g., as a light-activated tag. Covalent attachment of A heme moiety is generally aprosthetic group that consists of heme moiety an iron atom contained inthe center of a large heterocyclic organic ring, which is referred to asa porphyrin. The heme moiety may be used, e.g., as a tag for thepeptide. Attachment of a nucleotide May be used as a tag or as a basisfor further derivatising a or nucleotide derivative peptide.Cross-linking Cross-linking is a method of covalently joining twoproteins. Cross-linkers contain reactive ends to specific functionalgroups (primary amines, sulfhydryls, etc.) on proteins or othermolecules. Several chemical groups may be targets for reactions inproteins and peptides. For example, Ethylene glycolbis[succinimidylsuccinate, Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, and Bis[sulfosuccinimidyl]suberate link amines to amines. Cyclization For example, cyclization ofamino acids to create optimized delivery forms that are resistant to,e.g., aminopeptidases (e.g., formation of pyro glutamate, a cyclizedform of glutamic acid). Disulfide bond formation Disulfide bonds inproteins are formed by thiol-disulfide exchange reactions, particularlybetween cysteine residues (e.g., formation of cystine). DemethylationSee, e.g., U.S. Pat. No. 4,250,088 (Process for demethylating lignin).Formylation The addition of a formyl group to, e.g., the N-terminus of aprotein. See, e.g., U.S. Pat. Nos. 4,059,589, 4,801,742, and 6,350,902.Glycylation The covalent linkage of one to more than 40 glycine residuesto the tubulin C-terminal tail. Glycosylation Glycosylation may be usedto add saccharides (or polysaccharides) to the hydroxy oxygen atoms ofserine and threonine side chains (which is also known as O-linkedGlycosylation). Glycosylation may also be used to add saccharides (orpolysaccharides) to the amide nitrogen of asparagine side chains (whichis also known as N-linked Glycosylation), e.g., via oligosaccharyltransferase. GPI anchor formation The addition ofglycosylphosphatidylinositol to the C-terminus of a protein. GPI anchorformation involves the addition of a hydrophobic phosphatidylinositolgroup - linked through a carbohydrate containing linker (e.g.,glucosamine and mannose linked to phosphoryl ethanolamine residue) - tothe C-terminal amino acid of a protein. Hydroxylation Chemical processthat introduces one or more hydroxyl groups (—OH) into a protein (orradical). Hydroxylation reactions are typically catalyzed byhydroxylases. Proline is the principal residue to be hydroxylated inproteins, which occurs at the C^(γ) atom, forming hydroxyproline (Hyp).In some cases, proline may be hydroxylated at its C^(β) atom. Lysine mayalso be hydroxylated on its C^(δ) atom, forming hydroxylysine (Hyl).These three reactions are catalyzed by large, multi-subunit enzymesknown as prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl5-hydroxylase, respectively. These reactions require iron (as well asmolecular oxygen and α-ketoglutarate) to carry out the oxidation, anduse ascorbic acid to return the iron to its reduced state. IodinationSee, e.g., U.S. Pat. No. 6,303,326 for a disclosure of an enzyme that iscapable of iodinating proteins. U.S. Pat. No. 4,448,764 discloses, e.g.,a reagent that may be used to iodinate proteins. ISGylation Covalentlylinking a pep tide to the ISG15 (Interferon- Stimulated Gene 15)protein, for, e.g., modulating immune response. Methylation Reductivemethylation of protein amino acids with formaldehyde and sodiumcyanoborohydride has been shown to provide up to 25% yield ofN-cyanomethyl (—CH₂CN) product. The addition of metal ions, such asNi²⁺, which complex with free cyanide ions, improves reductivemethylation yields by suppressing by-product formation. TheN-cyanomethyl group itself, produced in good yield when cyanide ionreplaces cyanoborohydride, may have some value as a reversible modifierof amino groups in proteins. (Gidley et al.) Methylation may occur atthe arginine and lysine residues of a protein, as well as the N- andC-terminus thereof. Myristoylation Myristoylation involves the covalentattachment of a myristoyl group (a derivative of myristic acid), via anamide bond, to the alpha-amino group of an N-terminal glycine residue.This addition is catalyzed by the N-myristoyltransferase enzyme.Oxidation Oxidation of cysteines. Oxidation of N-terminal Serine orThreonine residues (followed by hydrazine or aminooxy condensations).Oxidation of glycosylations (followed by hydrazine or aminooxycondensations). Palmitoylation Palmitoylation is the attachment of fattyacids, such as palmitic acid, to cysteine residues of proteins.Palmitoylation increases the hydrophobicity of a protein.(Poly)glutamylation Polyglutamylation occurs at the glutamate residuesof a protein. Specifically, the gamma-carboxy group of a glutamate willform a peptide-like bond with the amino group of a free glutamate whosealpha-carboxy group may be extended into a polyglutamate chain. Theglutamylation reaction is catalyzed by a glutamylase enzyme (or removedby a deglutamylase enzyme). Polyglutamylation has been carried out atthe C- terminus of proteins to add up to about six glutamate residues.Using such a reaction, Tubulin and other proteins can be covalentlylinked to glutamic acid residues. Phosphopantetheinylation The additionof a 4′-phosphopantetheinyl group. Phosphorylation A process forphosphorylation of a protein or peptide by contacting a protein orpeptide with phosphoric acid in the presence of a non-aqueous apolarorganic solvent and contacting the resultant solution with a dehydratingagent is disclosed e.g., in U.S. Pat. No. 4,534,894. Insulin productsare described to be amenable to this process. See, e.g., U.S. Pat. No.4,534,894. Typically, phosphorylation occurs at the serine, threonine,and tyrosine residues of a protein. Prenylation Prenylation (orisoprenylation or lipidation) is the addition of hydrophobic moleculesto a protein. Protein prenylation involves the transfer of either afarnesyl (linear grouping of three isoprene units) or a geranyl-geranylmoiety to C-terminal cysteine(s) of the target protein. ProteolyticProcessing Processing, e.g., cleavage of a protein at a peptide bond.Selenoylation The exchange of, e.g., a sulfur atom in the peptide forselenium, using a selenium donor, such as selenophosphate. SulfationProcesses for sulfating hydroxyl moieties, particularly tertiary amines,are described in, e.g., U.S. Pat. No. 6,452,035. A process forsulphation of a protein or peptide by contacting the protein or peptidewith sulphuric acid in the presence of a non-aqueous apolar organicsolvent and contacting the resultant solution with a dehydrating agentis disclosed. Insulin products are described to be amenable to thisprocess. See, e.g., U.S. Pat. No. 4,534,894. SUMOylation Covalentlylinking a peptide a SUMO (small ubiquitin-related Modifier) protein,for, e.g., stabilizing the peptide. Transglutamination Covalentlylinking other protein(s) or chemical groups (e.g., PEG) via a bridge atglutamine residues tRNA-mediated addition of For example, thesite-specific modification (insertion) of an amino acids (e.g., aminoacid analog into a peptide. arginylation)

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are, accordingly, to be regarded in anillustrative rather than a restrictive sense.

The invention is illustrated herein by the experiments described aboveand by the following examples, which should not be construed aslimiting. The contents of all references, pending patent applicationsand published patents, cited throughout this application are herebyexpressly incorporated by reference. Those skilled in the art willunderstand that this invention may be embodied in many different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill fully convey the invention to those skilled in the art. Manymodifications and other embodiments of the invention will come to mindin one skilled in the art to which this invention pertains having thebenefit of the teachings presented in the foregoing description.Although specific terms are employed, they are used as in the art unlessotherwise indicated.

SEQUENCES

VLY protein from [organism = Strain 14018 and 14019 Gardnerellavaginalis] DNA Sequence ID NO: 1ATGAAGAGTACAAAGTTCTACCGTAATGCAGCAATGTTGCTCCTCGCGGGCGCAACTATTGTTCCACAATGCTTAGCAGCACCAGCAATGGCCGCTCCTTCCGCTAAGGATTCTGAACCAGCTACATCTTGCGCAGCTAAGAAAGACTCGTTGAATAATTATTTGTGGGATTTGCAATACGATAAAACAAACATTCTCGCCCGTCATGGCGAAACCATTGAGAACAAATTCTCCAGCGACAGCTTCAACAAGAACGGTGAATTCGTTGTTGTTGAGCATCAGAAGAAGAACATCACCAATACAACTTCAAATTTGTCGGTTACTTCCGCCAACGATGATCGCGTATACCCAGGTGCTCTTTTCCGTGCTGATAAGAATTTGATGGACAATATGCCAAGCCTGATTTCTGCAAACCGCGCTCCAATAACGTTGAGCGTTGATTTGCCGGGATTCCACGGCGGCGAAAGTGCTGTAACTGTTCAGCGCCCAACCAAGAGCTCTGTAACTTCCGCAGTGAACGGCTTAGTTTCTAAGTGGAATGCACAATATGGAGCAAGTCATCATGTTGCAGCTCGCATGCAGTACGATTCTGCAAGCGCACAAAGCATGAACCAGCTCAAGGCTAAGTTTGGTGCTGATTTTGCCAAGATTGGTGTTCCGCTGAAGATTGATTTCGATGCAGTACACAAGGGTGAGAAGCAGACTCAAATTGTGAACTTCAAGCAAACTTACTACACCGTAAGCGTTGATGCACCAGATAGCCCAGCAGATTTCTTTGCTCCTTGCACTACGCCAGACAGCTTGAAGAACCGTGGCGTTGACAACAAGCGCCCACCAGTTTACGTGTCAAACGTAGCTTATGGTCGCTCAATGTACGTAAAGTTCGATACCACCAGCAAGAGCACTGATTTCCAGGCTGCGGTAGAAGCAGCAATTAAGGGCGTAGAAATCAAGCCAAACACCGAATTCCATCGCATTCTCCAGAATACTTCTGTTACTGCAGTGATTCTTGGTGGCAGCGCTAATGGTGCAGCTAAAGTTATTACAGGCAATATCGATACGCTTAAGGCTTTGATTCAGGAAGGTGCAAATTTGAGCACCTCTAGCCCAGCGGTTCCAATTGCATACACCACTTCCTTCGTCAAGGATAACGAAGTAGCAACTTTGCAATCCAACAGCGATTATATTGAAACGAAGGTTTCTTCTTATCGCAATGGCTACTTGACTTTGGACCACCGTGGAGCTTATGTAGCTCGCTACTACATCTACTGGGATGAGTACGGCACCGAAATTGACGGCACTCCTTACGTGCGTTCTCGCGCTTGGGAAGGCAATGGTAAGTATCGTACAGCTCACTTCAACACCACTATTCAGTTCAAAGGAAATGTACGCAATCTACGAATCAAGTTGGTTGAAAAGACTGGTTTGGTTTGGGAACCATGGCGCACAGTATATGACCGTTCTGATTTGCCACTAGTTCGTCAGCGTACTATTAGCAACTGGGGCACAACCTTGTGGCCTCGCGTTGCTGAAACTGTAAAGAACGACTGAVL Y protein from [organism = Strain 14018 and 14019 Gardnerellavaginalis] Amino Acid Sequence ID NO: 2MKSTKFYRNAAMLLLAGATIVPQCLAAPAMAAPSAKDSEPATSCAAKKDSLNNYLWDLQYDKTNILARHGETIENKFSSDSFNKNGEFVVVEHQKKNITNTTSNLSVTSANDDRVYPGALFRADKNLMDNMPSLISANRAPITLSVDLPGFHGGESA VTVQRPTKSSVTSA VNGL VSKWNAQYGASHHV AARMQYDSASAQSMNQLKAKFGADF AKIGVPLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPDSLKNRGVDNKRPPVYVSNV A YGRSMYVKFDTTSKSTDFQAA VEAAIKGVEIKPNTEFHRILQNTSVTAVILGGSANGAAKVITGNIDTLKALIQEGANLSTSSPA VPIA YTTSFVKDNEV ATLQSNSDYIETKVSSYRNGYLTLDHRGA YV ARYYIYWDEYGTEIDGTPYVRSRA WEGNGKYRTAHFNTTIQFKGNVRNLRIKL VEKTGL VWEPWRTVYDRSDLPLVRQRTISNWGTTL WPRV AETVKND VLY protein from [organism =Gardnerella Vaginalis 49145] DNA SEQ ID NO: 3ATGAAGAGTACAAAGTTCTACCGTAATGCAGCAATGTTGCTCCTCGCGGGCGCAACTATTGTTCCACAATGCTTAGCAGCACCAGCAATGGCCGCTCCTTCCGCTAAGGATTCTGAACCAGCTACATCTTGCGCAGCTAAGAAAGACTCGTTGAATAATTATTTGTGGGATTTGCAATACGATAAAACAAACATTCTCGCCCGTCATGGCGAAACCATTGAGAACAAATTCTCCAGCGACAGCTTCAACAAGAACGGTGAATTCGTTGTTGTTGAGCATCAGAAGAAGAACATCACCAATACAACTTCAAATTTGTCGGTTACTTCCGCCAACGATGATCGCGTATACCCAGGTGCTCTTTTCCGTGCTGATAAGAATTTGATGGACAATATGCCAAGCCTGATTTCTGCAAACCGCGCTCCAATAACGTTGAGCGTTGATTTGCCGGGATTCCACGGCGGCGAAAGTGCTGTAACTGTTCAGCGCCCAACCAAGAGCTCTGTAACTTCCGCAGTGAACGGCTTAGTTTCTAAGTGGAATGCACAATATGGAGCAAGTCATCATGTTGCAGCTCGCATGCAGTACGATTCTGCAAGCGCACAAAGCATGAACCAGCTCAAGGCTAAGTTTGGTGCTGATTTTGCCAAGATTGGTGTTCCGCTGAAGATTGATTTCGATGCAGTACACAAGGGTGAGAAGCAGACTCAAATTGTGAACTTCAAGCAAACTTACTACACCGTAAGCGTTGATGCACCAGATAGCCCAGCAGATTTCTTTGCTCCTTGCACTACGCCAGACAGCTTGAAGAACCGTGGCGTTGACAACAAGCGCCCACCAGTTTACGTGTCAAACGTAGCTTATGGTCGCTCAATGTACGTAAAGTTCGATACCACCAGCAAGAGCACTGATTTCCAGGCTGCAGTAGAAGCAGCAATTAAGGGCGTAGAAATCAAGCCAAACACCGAATTCCATCGCATTCTCCAAAATACTTCTGTTACTGCAGTGATTCTTGGTGGCAGCGCTAATGGTGCAGCTAAAGTTATTACAGGCAACATCGATACGTTGAAGGCTTTGATTCAGGAAGGTGCAAATTTGAGCACCTCTAGCCCAGCAGTTCCAATTGCATACACCACTTCCTTCGTCAAGGATAACGAAGTAGCAACTTTGCAATCCAACAGCGATTATATTGAAACGAAGGTTTCCTCTTACCGCAATGGCTACTTGACTTTGGACCACCGTGGAGCTTACGTAGCTCGCTACTACATCTACTGGGATGAGTACGGCACCGAAATTGACGGCACTCCTTACGTGCGTTCTCGCGCTTGGGAAGGCAATGGTAAGTATCGTACAGCTCACTTCAATACCACTATTCAGTTCAAAGGAAATGTACGCAATCTACGAATCAAGTTGGTTGAAAAGACTGGTTTAGTTTGGGAACCATGGCGCACAGTATATGACCGTTCTGATTTGCCACTAGTTCATCAGCGTACTATTAGCAACTGGGGCACAACCTTGTGGCCTCGCGTTGCTGAAACTGTAAAGAACGACTGAVLY DOMAIN 4 DNA FROM Gardnerella vaginalis 14018 and 14019 nucleicacid 1126 to the end 1551 V SEQ ID NO: 4  TACACCACTTCCTTCGTCAAGGATAACGAAGTAGCAACTTTGCAATCCAACAGCGATTATATTGAAACGAAGGTTTCTTCTTATCGCAATGGCTACTTGACTTTGGACCACCGTGGAGCTTATGTAGCTCGCTACTACATCTACTGGGATGAGTACGGCACCGAAATTGACGGCACTCCTTACGTGCGTTCTCGCGCTTGGGAAGGCAATGGTAAGTATCGTACAGCTCACTTCAACACCACTATTCAGTTCAAAGGAAATGTACGCAATCTACGAATCAAGTTGGTTGAAAAGACTGGTTTGGTTTGGGAACCATGGCGCACAGTATATGACCGTTCTGATTTGCCACTAGTTCGTCAGCGTACTATTAGCAACTGGGGCACAACCTTGTGGCCTCGCGTTGCTGAAACTGTAAAGAACGACTGAVLY DOMAIN 4 DNA FROM Gardnerella vaginalis 49145 nucleic acid 1126to the end 1551 V SEQ ID NO: 12TACACCACTTCCTTCGTCAAGGATAACGAAGTAGCAACTTTGCAATCCAACAGCGATTATATTGAAACGAAGGTTTCCTCTTACCGCAATGGCTACTTGACTTTGGACCACCGTGGAGCTTACGTAGCTCGCTACTACATCTACTGGGATGAGTACGGCACCGAAATTGACGGCACTCCTTACGTGCGTTCTCGCGCTTGGGAAGGCAATGGTAAGTATCGTACAGCTCACTTCAATACCACTATTCAGTTCAAAGGAAATGTACGCAATCTACGAATCAAGTTGGTTGAAAAGACTGGTTTAGTTTGGGAACCATGGCGCACAGTATATGACCGTTCTGATTTGCCACTAGTTCATCAGCGTACTATTAGCAACTGGGGCACAACCTTGTGGCCTCGCGTTGCTGAAACTGTAAAGAACGACTGA VLYDOMAIN 4Amino Acid FROM Gardnerella vaginalis 14018 and 14019 SEQ ID NO: 5YTTSFVKDNEV ATLQSNSDYIETKVSSYRNGYLTLDHRGA YV ARYYIYWDEYGTEIDGTPYVRSRA WEGNGKYRTAHFNTTIQFKGNVRNLRIKL VEKTGL VWEPWRTVYDRSDLPL VRQRTISNWGTTLWPRV AETVKND VLYDOMAIN 4 Amino AcidFROM Gardnerella vaginalis 49145: SEQ ID NO: 13YTTSFVKDNEV ATLQSNSDYIETKVSSYRNGYLTLDHRGA YV ARYYIYWDEYGTEIDGTPYVRSRA WEGNGKYRTAHFNTTIQFKGNVRNLRIKL VEKTGL VWEPWRTVYDRSDLPL VHQRTISNWGTTL WPRV AETVKND VLY UndecapeptideFROM Gardnerella vaginalis 14018 and 14019nucleic acid residues 1414-1446 DNA SEQ ID NO: 6GAAAAGACTGGTTTGGTTTGGGAACCATGGCGC VLY UndecapeptideFROM Gardnerella vaginalis 49145 nucleic acid residues 1414-1446DNA SEQ ID NO: 14 GAAAAGACTGGTTTAGTTTGGGAACCATGGCGC VLY UNDECAPEPTIDEFROM Gardnerella vaginalis (CONSERVED)Amino acids 472 to 482 AMINO ACID SEQUENCE SEQ ID NO: 7 EKTGLVWEPWRVLY UNDECAPEPTIDE P480W TOXOID FROM Gardnerella vaginalis (CONSERVED)Undecapeptide amino acids 472 to 482 Amino Acid SEQ ID NO: 8 EKTGLVWEWWRCodon optimized VLY gene sequence (same protein sequence as VLYfrom strain 14018) DNA SEQ ID NO: 9ATGAAAAGCACCAAATTTTATCGTAACGCGGCGATGCTGCTGCTGGCAGGTGCAACCATTGTGCCGCAGTGCCTGGCAGCACCGGCAATGGCAGCACCGAGCGCAAAAGATAGCGAACCGGCGACCAGCTGCGCGGCGAAAAAAGATAGCCTGAACAACTATCTGTGGGATCTGCAGTATGATAAAACCAACATTCTGGCGCGTCATGGCGAAACCATTGAAAACAAATTTAGCAGCGATAGCTTTAACAAAAACGGCGAATTTGTGGTGGTGGAACATCAGAAGAAAAACATTACCAACACCACCAGCAACCTGAGCGTGACCAGCGCGAACGATGATCGTGTGTATCCGGGCGCGCTGTTTCGTGCGGATAAAAACCTGATGGATAACATGCCGAGCCTGATTAGCGCGAACCGTGCGCCGATTACCCTGAGCGTGGATCTGCCGGGCTTTCATGGCGGCGAAAGCGCGGTGACCGTGCAGCGTCCGACCAAAAGCAGCGTGACCAGCGCGGTGAACGGCCTGGTTAGCAAATGGAACGCGCAGTATGGCGCGAGCCATCATGTGGCGGCGCGTATGCAGTATGATAGCGCGAGCGCGCAGAGCATGAACCAGCTGAAAGCGAAATTTGGCGCGGATTTTGCGAAAATTGGCGTGCCGCTGAAAATTGATTTTGATGCGGTGCATAAAGGCGAAAAACAGACCCAGATTGTGAACTTTAAACAGACCTATTATACCGTGAGCGTGGATGCGCCGGATAGCCCGGCGGATTTCTTTGCGCCGTGCACCACCCCGGATAGCCTGAAAAACCGTGGCGTGGATAACAAACGTCCGCCGGTGTATGTGAGCAACGTGGCGTATGGCCGTAGCATGTATGTGAAATTTGATACCACCAGCAAAAGCACCGATTTTCAGGCGGCGGTGGAAGCGGCGATTAAAGGCGTGGAAATTAAACCGAACACCGAATTTCATCGTATTCTGCAGAACACCAGCGTGACCGCGGTGATTCTGGGCGGCAGCGCGAACGGCGCGGCGAAAGTGATTACCGGCAACATTGATACCCTGAAAGCGCTGATTCAGGAAGGCGCGAACCTGAGCACCAGCAGCCCGGCGGTGCCGATTGCGTATACCACCAGCTTTGTGAAAGATAACGAAGTGGCGACCCTGCAGAGCAACAGCGATTATATTGAAACCAAAGTGAGCAGCTATCGTAACGGCTATCTGACCCTGGATCATCGTGGCGCGTATGTGGCGCGTTATTATATTTATTGGGATGAATATGGCACCGAAATTGATGGCACCCCGTATGTGCGTAGCCGTGCGTGGGAAGGCAACGGCAAATATCGTACCGCGCATTTTAACACCACCATTCAGTTTAAAGGCAACGTGCGTAACCTGCGTATTAAACTGGTGGAAAAAACCGGCCTGGTGTGGGAACCGTGGCGTACCGTGTATGATCGTAGCGATCTGCCGCTGGTGCGTCAGCGTACCATTAGCAACTGGGGCACCACCCTGTGGCCGCGTGTGGCGGAAACCGTGAAAAACGATTAAGardnerella vaginalis strain ARG3 VLY DNA SEQ ID NO: 10ATGAAGAGTACAAAGTTCTACCGTAATGCAGCAATGTTGCTCCTCGCGGGCGCAACTATTGTTCCACAATGCTTAGCAGCACCAGCAATGGCCGCTCCTTCCGCTAAGGATTCTGAACCAGCTACATCTTGCGCAGCTAAGAAAGACTCGTTGAATAATTATTTGTGGGATTTGCAATACGATAAAACAAACATTCTCGCCCGTCATGGCGAAACCATTGAGAACAAATTCTCCAGCGACAGCTTCAACAAGAACGGTGAATTCGTTGTTGTTGAGCATCAGAAGAAGAACATCACCAATACAACTTCAAATTTGTCGGTTACTTCCGCCAACGATGATCGCGTATACCCAGGTGCTCTTTTCCGTGCTGATAAGAATTTGATGGACAATATGCCAAGCCTGATTTCTGCAAACCGCGCTCCAATAACGTTGAGCGTTGATTTGCCGGGATTCCACGGCGGCGAAAGTGCTGTAACTGTTCAGCGCCCAACCAAGAGCTCTGTAACTTCCGCAGTGAACGGCTTAGTTTCTAAGTGGAATGCACAATATGGAGCAAGTCATCATGTTGCAGCTCGCATGCAGTACGATTCTGCAAGCGCACAAAGCATGAACCAGCTCAAGGCTAAGTTTGGTGCTGATTTTGCCAAGATTGGTGTTCCGCTGAAGATTGATTTCGATGCAGTACACAAGGGTGAGAAGCAGACTCAAATTGTGAACTTCAAGCAAACTTACTACACCGTAAGCGTTGATGCACCAGATAGCCCAGCAGATTTCTTTGCTCCTTGCACTACGCCAGACAGCTTGAAGAACCGTGGCGTTGACAACAAGCGCCCACCAGTTTACGTGTCAAACGTAGCTTATGGTCGCTCAATGTACGTAAAGTTCGATACCACCAGCAAGAGCACTGATTTCCAGGCTGCGGTAGAAGCAGCAATTAAGGGCGTAGAAATCAAGCCAAACACCGAATTCCATCGCATTCTCCAGAATACTTCTGTTACTGCAGTGATTCTTGGTGGCAGCGCTAATGGTGCAGCTAAAGTTATTACAGGCAATATCGATACGCTTAAGGCTTTGATTCAGGAAGGTGCAAATTTGAGCACCTCTAGCCCAGCGGTTCCAATTGCATACACCACTTCCTTCGTCAAGGATAACGAAGTAGCAACTTTGCAATCCAACAGCGATTATATTGAAACGAAGGTTTCTTCTTATCGCAATGGCTACTTGACTTTGGACCACCGTGGAGCTTATGTAGCTCGCTACTACATCTACTGGGATGAGTACGGCACCGAAATTGACGGCACTCCTTACGTGCGTTCTCGCGCTTGGGAAGGCAATGGTAAGTATCGTACAGCTCACTTCAACACCACTATTCAGTTCAAAGGAAATGTACGCAATCTACGAATCAAGTTGGTTGAAAAGACTGGTTTGGTTTGGGAACCATGGCGCACAGTATATGACCGTTCTGATTTGCCACTAGTTCGTCAGCGTACTATTAGCAATTGGGGCACAACCTTGTGGCCTCGCGTTGCTGAAACTG TAAAGAACGACTGAARG3 VLY protein sequence SEQ ID NO. 11MKSTKFYRNAAMLLLAGATIVPQCLAAPAMAAPSAKDSEPATSCAAKKDSLNNYLWDLQYDKTNILARHGETIENKFSSDSFNKNGEFVVVEHQKKNITNTTSNLSVTSANDDRVYPGALFRADKNLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVQRPTKSSVTSAVNGLVSKWNAQYGASHHVAARMQYDSASAQSMNQLKAKFGADFAKIGVPLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPDSLKNRGVDNKRPPVYVSNVAYGRSMYVKFDTTSKSTDFQAAVEAAIKGVEIKPNTEFHRILQNTSVTAVILGGSANGAAKVITGNIDTLKALIQEGANLSTSSPAVPIAYTTSFVKDNEVATLQSNSDYIETKVSSYRNGYLTLDHRGAYVARYYIYWDEYGTEIDGTPYVRSRAWEGNGKYRTAHFNTTIQFKGNVRNLRIKLVEKTGLVWEPWRTVYDRSDLPLVRQRTISNWGTTLWPRVAETVKND*

EXAMPLES Example 1 Materials and Methods

Bacterial Strains and Cell Lines

Gardnerella vaginalis strains 14018, 14019, and 49145 were obtained fromATCC. ARG3 is a clinical isolate of G. vaginalis kindly provided bySusan Whittier. Cells were grown in brain-heart infusion supplementedwith 5% fetal bovine serum, 0.3% Tween 80, and 0.1% soluble starch or in10% fetal bovine serum (HyClone), 5% Fildes enrichment (Remel) and 4ng/ml of amphotericin. There are many efficient ways to cultureGardnerella vaginalis known in the art. Cultures were typicallyincubated at 37° C. and 5% CO₂ . E. coli strains TOP10 and BL21AI(Invitrogen) were grown in LB, with kanamycin (30 μg/ml) selection asappropriate. HeLa cells were grown at 37° C./5% CO₂ in MEM supplementedwith 10% fetal bovine serum and 10 μg/ml ciprofloxacin. CHO-K1 cells(CCL-61) were grown at 37° C./5% CO₂ in F12 Kaighn's Modification(Invitrogen) with 10% FBS and 10 μg/ml ciprofloxacin.

Human cervical endothelial cells (HeLa, ATCC CCL-2) were grown at 37° C.and 5% CO₂ in minimal essential medium (Invitrogen) supplemented with10% fetal bovine serum and 10 μg/ml ciprofloxacin. Human vaginalendothelial cells (VK2, ATCC CRL-2616) were grown in serum freekeratinocyte growth media (Invitrogen) with 0.1 ng/ml EGF, 0.05 mg/mlbovine pituitary extract and 0.4 mM calcium chloride (Biol Reprod 1997,October 57(4):847-55, Generation of papillomavirus-immortalized celllines from normal human ectocervical, endocervical, and vaginalepithelium that maintain expression of tissue-specific differentiationproteins; Fichorova R N, Rheinwald J G, Anderson D J).

Cloning, Sequencing, and Analysis of the VLY Gene

The G. vaginalis genomic region containing VLY was amplified from G.vaginalis 14018 by PCR using Pfx proofreading polymerase (Invitrogen)and primers V1(ATGCAGCGAAGCATGCCATGC) (SEQ ID NO: 15) andV2(TCAGTCGTTCTTTACAGTTTC) (SEQ ID NO: 16). This PCR product was clonedinto vector pCR2.1/TOPO (Invitrogen) and transformed into E. coli TOP10according to the manufacturer's instructions. (50) The insert wasbidirectionally sequenced using vector-specific primers. The predictedVLY open reading frame was amplified by PCR using the cloned genomicregion as template, Pfx polymerase, and primersV3(GCCGCCGCCCATATGAAGAGTACAAAG) (SEQ ID NO: 17) andV6(GCCGGATCCTCAGTCGTTCTTTACAGT) (SEQ ID NO: 18), adding uniquerestriction sites indicated by underlining. The resulting product wascut with restriction enzymes Ndel and BamHI, cloned into the vectorpET28a (Novagen) to generate a construct with an N-terminalhexahistidine transcriptional fusion, and confirmed by sequencing.Site-directed mutagenesis to construct (pET28a/VLY(P480W)) was performedwith the QuikChange II XL kit (Stratagene) according to themanufacturer's instructions. Mutagenic primers used were P480Wsense(TGGTTGAAAAGACTGGTTTGGTTTGGGAATGGTGGCGCACAGTATAT) (SEQ ID NO: 19) andP480Wanti (ATATACTGTGCGCCACCATTCCCAAACCAAACCAGTCTTTTCAACCA) (SEQ ID NO:20).

Improved purity and greater yield were achieved by generating atruncated construct (excluding the first 50 amino acids from theN-terminal region) using the primer VLY50up(5′-GCCGCCCATATGTCGTTGAATAATTATTTGTGG-3′) (SEQ ID NO: 21) along with thepreviously described V6 primer. The PCR product was cloned into thepET28a vector (Novagen), confirmed by sequencing, and transformed intoE. coli BL21-AI competent cells (Invitrogen) for expression andpurification as previously described. ¹¹ The lytic activity of thistruncated recombinant toxoid was unaltered (data not shown). The DNAsequence encoding VLY protein is set forth in SEQ ID NO: 1 for strains14018 an 14019; the amino acid sequence encoding VLY protein is setforth in SEQ ID NO:2.

Protein sequence prediction, alignment, and phylogenetic analyses wereperformed using MacVector software (Version 9.5, MacVector Inc.).Protein sequences for other CDC family members (Appendix) were obtainedfrom the Comprehensive Microbial Resource (J. Craig Venter Institute,http://cmr.tigr.org) or from the GenBank/Entrez Protein database(National Center for Biotechnology Information).

Expression and Purification of Recombinant Toxins

E. coli BL21AI carrying the pET28a/VLY or pET28a/VLY(P480W) plasmid weregrown in one liter cultures at 37° C. on a rotary shaker for 3 hr, andprotein expression was induced with 1 mM IPTG and 0.02% L-arabinose(Sigma). After 6 hr, bacteria were pelleted, lysed with BugBustersolution (Novagen) in the presence of protease inhibitor cocktail,lysozyme (100 μg/ml), and benzonase nuclease, all from Sigma. Lysateswere cleared by centrifugation and tagged recombinant toxin purifiedusing Ni-NTA agarose (Qiagen) according to the manufacturer'sinstructions. Purified toxin was extensively dialysed against LPS-freePBS (Gibco) to remove imidazole and concentrated (Amicon Ultra, 10 kD MWcutoff). Protein concentrations were determined using a modifiedBradford assay (Bio-Rad).

The coding sequence of the pneumolysin gene was amplified by PCR usingprimers Ndel-Ply-up (GGAATTCCATATGGCAAATAAAGCAG) (SEQ ID NO: 22) andPly-down-Xhol (CCGCTCGAGGTCATTTTCTACCTTATC) (SEQ ID NO: 23) usinggenomic DNA of S. pneumoniae strain TIGR4 as a template. These primersadded unique restriction sites as indicated by underlining and led toamplification of the entire pneumolysin sequence, omitting the stopcodon to allow addition of a C-terminal hexahistidine taG. The productwas confirmed by sequencing, digested with Ndel and Xhol (New EnglandBiolabs), and cloned into pET29a (Novagen) cut with Ndel and Xhol. Theplasmid was transformed into E. coli BL21-AI, and induction andpurification were performed as for VLY. Site-directed mutagenesis usedprimers W435Psense (ACCGGGCTTGCCTGGGAACCGTGGCGTACG) (SEQ ID NO: 24) andW435Panti (CGTACGCCACGGTTCCCAGGCAAGCCCGGT) (SEQ ID NO: 25).

Anti-pneumolysin Western Blot

G. vaginalis 14018 was grown on chocolate agar and fresh coloniesscraped from the plate and resuspended in lysis buffer (BugBuster, EMDChemicals, Gibbstown, N.J.) with benzonase nuclease. The lysate wasboiled for 5 minutes, and 30 μl of lysate separated on a 4-12%polyacrylamide gel (Invitrogen). Purified VLY (500 ng total) was run asa positive control. The proteins were transferred to PVDF membranes,blocked with 5% milk, and probed with murine monoclonal anti-pneumolysin(clone 9.1/2/3/6; Novocastra, Newcastle Upon Tyne, UK; 1:100 dilution).Detection was with HRP-conjugated anti-mouse IgG (Santa CruzBiotechnology, Santa Cruz, Calif.) and ECL.

Anti-VLY Western Blot

G. vaginalis 14018 was grown on an HBT plate and fresh colonies wereresuspended in lysis buffer (BugBuster, EMD Chemicals, Gibbstown N.J.)with benzonase nuclease. The lysate was boiled and separated on a 10%polyacrylamide gel. Proteins were transferred to polyvinylidenedifluoride membranes, blocked with 5% milk and probed using rabbitpolyclonal anti-VLY antiserum (1:500,000 dilution). Detection was withHRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) and ECL.Membranes probed with pre-immune serum served as a negative control.

Erythrocyte Lysis Assay

The use of human erythrocytes was approved by the Columbia UniversityInstitutional Review Board. Human blood was obtained by venipuncture anderythrocytes immediately isolated by centrifugation and repeated washingin sterile PBS. For human samples, a 1% solution of packed erythrocytesin sterile PBS was prepared and added to a 96-well polystyreneV-bottomed plate (100 μl/well). Blood from other species tested wasobtained commercially (Fisher Scientific) and erythrocytes washed insterile PBS prior to use. A 1% solution of packed erythrocytes insterile PBS was combined with an equal volume of toxin diluted in PBS.The total volume for the assay was 200 μl per well of a 96-wellpolystyrene V-bottom plate. The negative control for lysis consisted ofPBS without toxin added to erythrocytes, and the positive control for100% lysis was 0.05% Triton X-100. Incubation was for 30 min at 37°C./5% CO₂. At the conclusion of the assay, the plates were spun at 2000rpm to pellet erythrocytes and supernatants removed for measurement ofoptical density at 415 nm. Where noted, toxins were preincubated withcholesterol (stock solution 100 mg/ml in chloroform; workingconcentration 1-10 μg/ml) or control (chloroform alone at thecorresponding dilution) for 10 min at room temperature prior to use inthe assay. Antibody inhibition experiments were performed usinganti-CD55 (clone IA10, BD Pharmingen), anti-CD59 (clone YTH53.1,GeneTex), or irrelevant antibody control. Preincubation of erythrocyteswith antibody (9 ng/ml final concentration) was for 1 hr at 4° C. withconstant rotation, followed by two PBS washes to remove unbound antibodyprior to use in the assay.

CHO Cell Transfection and LDH Release Assay

The coding sequence for human CD59 was amplified from cDNA fromA549(CCL-185) respiratory epithelial cells using primersCD59-1(GCCGCCCTCGAGCCACCAATGGGAATCCAAGGAG) (SEQ ID NO: 26) and CD59-2(GCCGCCGAATTCTTAGGGATGAAGGCTCCAGGC) (SEQ ID NO: 27) and cloned into theXhol and EcoRI sites of pIRES2-EGFP (Clontech). Sequence was confirmedusing vector specific primers. CHO-K1 cells were transfected withpurified plasmid DNA (either pIRES2-EGFP/CD59 or the corresponding emptyvector control) using a Nucleofector (Amaxa) according to themanufacturer's instructions. Transfected cells were plated into 6-welldishes and used 48 hr after transfection. Greater than 90% transfectionefficiency was assessed by fluorescence microscopy (data not shown).Cells were weaned from serum overnight and stimulated with VLY orpneumolysin diluted in serum-free F12 media for 30 min at 37° C. / 5%CO₂. Cell viability was confirmed at the end of the experiment by visualinspection of the monolayer and trypan blue exclusion and exceeded 90%.The positive control for complete lysis was 1% Triton X-100 inserum-free F12. The concentration of lactate dehydrogenase insupernatants was assessed with a commercial kit (Roche) according to themanufacturer's instructions.

Epithelial p38 MAPK Phosphorylation

Western blot analysis of epithelial p38 MAPK phosphorylation wasperformed as previously described (32).

Real-time PCR

HeLa cells were weaned from serum overnight and treated for 2 hr withmedium alone, VLY (10 μg/ml), or VLY(P480W) (10μg/ml). Cells were lysedin RLT+buffer (Qiagen) and RNA purified using a commercially availablekit (RNeasy Plus; Qiagen). Reverse transcription of 1.5 μg of RNA persample to generate cDNA was with the high-capacity cDNA kit (AppliedBiosystems). Real-time PCR (Applied Biosystems StepOne) with SYBR greendetection (PowerSYBR, Applied Biosystems) was performed using primersfor interleukin-8 (TACTCCAAACCTTTCCAACCC and AACTTCTCCACAACCCTCTG) (SEQID NOS: 28 and 29) and GAPDH (GGGCGCCTGGTCACCAGGGCTG andGGGGCCATCCACAGTCTTCTG) (SEQ ID NOS: 30 and 31). Relative quantitationused the ΔΔC_(T) method with normalization to GAPDH.

Generation of Antibodies

Purified VLY toxin was generated and submitted to Cocalico Biologicals,Inc. (Reamstown, Pa.). According to their protocol, adult rabbits wereinjected with a minimum of 100 μg antigen mixed with Complete Freund'sAdjuvant subcutaneous and/or intramuscularly at multiple sites. Boosterdoses containing a minimum of 50 μg antigen mixed with IncompleteFreund's Adjuvant were administered on days 14, 21 and 49. A test bleedwas performed on day 56. Prior to the first immunization, serum wascollected from each rabbit to serve as negative control.

Immunofluorescence

G. vaginalis 14018 was grown to in culture media and bacterial cellswere fixed on a glass chamber slide using 4% paraformaldehyde.Non-specific binding sites were blocked using 5% normal donkey serum and0.2% triton X-100. Pre-immune or immune serum was added to each slide(1:500 dilution) for 1 h at room temperature. Following serial washeswith PBS and 0.2% triton X-100, donkey anti-rabbit conjugated to AlexaFluor (AF)-488 (Invitrogen; 1:1000 dilution) was added for 30 min in thedark with gentle shaking. After washing, chambers were removed from theslide and cover slips were mounted with ProLong Gold antifade with DAPI(Invitrogen). Slides to which no primary antibody was added served asnegative controls.

ELISA Based Assay for VLY Production

Four strains of G. vaginalis (14018, 14019, 49145, and ARG3) were grownon HBT plates, colonies were scraped and inoculated into 30 ml of liquidmedia. A 500 μl aliquot of each culture was obtained every 6 hours fordetermination of OD₆₀₀. An additional 1 ml sample from each was pelletedby centrifugation and supernatant stored at −20° C. prior to ELISA.

Immuno-96 MicroWell plates (Nunc) were coated with anti-pneumolysinantibody (clone 1F11, previously shown to cross-react with VLY) diluted1:500 in coating buffer (0.1 M sodium carbonate, pH: 9.5) and incubatedat 4° C. overnight. Wells were washed with PBS and 0.05% Tween 20.Non-specific binding sites were blocked using PBS with 10% fetal bovineserum for 1 h. Supernatants (100 μl) were added to each well and plateswere incubated at room temperature for 2 h. Known concentrations ofrecombinant VLY toxin diluted in G. vaginalis culture media were used asstandards. Rabbit polyclonal anti-VLY antiserum (diluted 1:1000 inblocking solution) was added to each well for 30 min at roomtemperature. After washing, goat anti-rabbit HRP antibody (Santa Cruzbiotechnology Inc., 1:1000 dilution) was added for 30 min. Wells werethoroughly washed and 100 μl of TMB substrate (Thermo Scientific) wasadded to each well and plate was incubated in the dark for 15 min. 50 μlof stop solution (2N sulfuric acid) was added to each well and OD₄₅₀determined.

Cytotoxicity Assay

24-well plates were seeded with VK2 or HeLa human epithelial cells inappropriate media and grown to >90% confluence. 12 hours prior to use,HeLa cells were weaned from serum. Recombinant VLY toxin diluted inmedia (10 μg/ml) or vehicle control was added to each well. Whereindicated, toxin was preincubated with pre-immune or immune sera for 30min at 4° C. prior to use in the assay. The plates were incubated for 45min at 37° C. and 5% CO₂. Supernatant was removed and the concentrationof lactate dehydrogenase was determined using a commercial kit (Roche)per the manufacturer's instructions.

Statistical Analysis

Statistical comparisons were performed using two-tailed unpaired t-testsor one-way analysis of variance (ANOVA) with Tukey post-test asappropriate (Prism, GraphPad Software).

Sequence Data Availability

The sequence data for the G. vaginalis genome for strains 14018, 14019,and 49145 are available in GenBank under the accession numbersEU522486-EU522488.

Example 2

Domain 4 of VLY Plays a Role in Species Specificity

Consistent with these findings, the hCD59 binding site has beenlocalized to this domain for ILY (22) and for VLY (FIG. 8 renumber).Using overlap-extension PCR, we generated a toxin chimera, containingdomains 1-3 of VLY and domain 4 of PLY, a species non-selective CDC(FIG. 8A). Unlike the parent VLY, the chimera lysed human and non-humanerythrocytes with equal efficacy (FIG. 8B), and does not require hCD59for CHO cell lysis (FIG. 8C). This indicates that D4 plays a major rolein species-selectivity among the CDCs. In order to generate a probe fortoxin-hCD59 interactions, we created a GFP:VLYD4 fusion protein (FIG.8D). This protein binds to hCD59-expressing human epithelial cells butdoes not form pores (FIG. 8E). Thus, this protein will be an invaluabletool for studies delineating requirements for binding between D4 andhCD59.

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December 1999;180(6):1863-1868.

What is claimed is:
 1. A method for diagnosing and treating a G.vaginalis infection or bacterial vaginosis in a patient, comprising: a.obtaining a biological sample from the patient, b. detecting thepresence of vaginolysin protein having the amino acid sequence set forthin SEQ ID NO: 2 or SEQ ID NO: 11, or an immunogenic fragment thereof inthe biological sample using a sandwich ELISA assay in which soluble CD59is used as a capture agent and one of an anti-vaginolysin antibodyincluding poly anti-vaginolysin, anti-pneumolysin antibody, monoclonalanti-vaginolysin and anti-intermedialysin is used as a detectionantibody, c. and if the respective protein is detected thenadministering to the patient a therapeutically effective amount of anantibiotic that is selected from the group consisting of metronidazole,clindamycin, and tinidazole, or a protective agent that is a memberselected from the group consisting of anti-vaginolysin antibody,anti-pneumolysin antibody, anti-CD59 antibody, soluble CD59, G.vaginalis vaginolysin toxoid, or an immunogenic fragment thereof.